Synthetic T cell receptor antigen receptor that specifically binds LILRB4 and uses thereof

Abstract

The present invention discloses a synthetic T cell receptor antigen receptor that specifically binds LILRB4 and its use. The present invention aims to obtain a more effective treatment against tumors by modifying TCR and CAR with an antigen-binding fragment that targets LILRB4.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and in particular to synthetic T cell receptor antigen receptors (STARs) that specifically bind LILRB4, STAR complexes, immune cells comprising STARs or STAR complexes, and their uses in the biomedical field. [Background technology]

[0002] LILRB4 is a member of the leukocyte immunoglobulin receptor family, possessing a single transmembrane domain, two extracellular C-type Ig-like domains, and three intracellular tyrosine-based immunoreceptor inhibitory motifs (ITIMs). In normal humans, LILRB4 is expressed primarily in myeloid immune cells, such as macrophages, dendritic cells (DCs), and monocytes, where it plays an immunosuppressive role. LILRB4 is highly expressed in acute myelomonocytic leukemia and acute monocytic leukemia, with expression levels higher than those of normal monocytes. LILRB4 expression is also detected in leukemic tumor stem cells. It is also highly expressed in various immunosuppressive cells in the tumor microenvironment, such as tumor adenocarcinoma (TAM), myeloblastoma-like stem cells (M-MDSCs), tolerogenic DCs, and Tregs. Studies have shown that LILRB4 expressed in AML promotes tumor cell migration and invasion and inhibits T cell proliferation through arginase-1 and uPAR, while its expression in other bone marrow immune cells, such as macrophages, dendritic cells (DCs), and monocytes, significantly promotes immune cell differentiation toward immune tolerance, thereby exerting immunosuppressive effects. Based on its specific expression in leukemia cells and bone marrow immune cells and its immunosuppressive function, LILRB4 may be an ideal therapeutic target for acute myeloid monocytic leukemia and acute monocytic leukemia by eliminating tumor cells and improving the immunosuppressive environment.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy has achieved promising results in recent years as an anti-cancer immunotherapy. Unlike the way natural T cells recognize tumor cells, CAR-T cells recognize tumor cells independently of MHC molecules. CAR molecules contain three parts: an extracellular region, which is an antigen recognition domain derived from antibodies and is responsible for recognizing target antigens; a transmembrane region; and an intracellular region, which contains signaling molecules and costimulatory signaling molecules derived from T cell receptors and is responsible for transmitting T cell activation signals after stimulation. The mechanism of action is as follows: when CAR molecules bind to their corresponding antigens, they aggregate, thereby increasing local phosphorylation levels and activating downstream signals, ultimately initiating the effector function of T cells and killing the target tumor cells.

[0004] The T cell receptor (TCR) complex contains multiple chains. The TCRα and TCRβ chains are responsible for recognizing MHC peptide molecules, while the other six CD3 subunits bind to the TCRα / β chains to perform signaling functions. The natural TCR complex contains a total of 10 ITAM signal sequences, which theoretically could transmit a stronger signal than CARs. Previous studies have shown that TCR signals are transmitted more slowly than CAR signals, but are more persistent. Therefore, by utilizing the signaling functions of natural TCRs, it may be possible to construct new receptors that can alleviate T cell dysfunction and better enable T cells to perform their anti-solid tumor role.

[0005] Because the extracellular region of the TCR is highly similar to the Fab domain of an antibody, replacing the TCR variable region sequence with an antibody variable region sequence can yield a synthetic T cell receptor antigen receptor (STAR) that combines the specificity of an antibody with the superior signaling function of a natural TCR, allowing it to mediate full T cell activation.

[0006] However, STARs derived from natural TCRs still suffer from drawbacks such as low membrane stability, low α / β chain pairing ability, mismatches with endogenous TCRs, and difficulty in introduction into T cells. Therefore, there remains a need in the art for improved STARs. Summary of the Invention [Problem to be solved by the invention]

[0007] To address the deficiencies of the prior art, the present invention provides a synthetic T cell receptor (antigen receptor STAR) that specifically binds LILRB4 and can modify TCRs and CARs with antigen-binding fragments that target LILRB4, and uses thereof. Existing results support that the STAR-T cells of the present invention are highly promising for the treatment of relapsed and refractory AML FAB M4 / M5, with therapeutic efficacy and safety superior to conventional technologies such as CAR-T. Based on the high expression of LILRB4 in AML FAB M4 / M5 and its high expression in bone marrow immunosuppressive cells, the development of LILRB4 dual-epitope STAR-T may provide a new immune cell therapy strategy for R / R AML FAB M4 / M5 patients through tumor cell killing and improvement of the tumor microenvironment.

[0008] Specifically, in a first aspect of the invention there is provided a synthetic T cell receptor antigen receptor (STAR), comprising: i) the synthetic T cell receptor antigen receptor comprises an alpha chain and a beta chain, wherein the alpha chain comprises a first target binding region and a first constant region, and the beta chain comprises a second target binding region and a second constant region, or wherein the alpha chain comprises a first target binding region and the beta chain comprises a second target binding region and a second constant region, or ii) The synthetic T cell receptor antigen receptor comprises a gamma chain and a delta chain, wherein the gamma chain comprises a first target binding region and a first constant region, and the delta chain comprises a second target binding region and a second constant region; or the gamma chain comprises a first target binding region and a first constant region, and the delta chain comprises a second target binding region and a second constant region.

[0009] Preferably, i) the α chain and / or the β chain have at least one functional domain linked to their C-terminus, or ii) the γ chain and / or the δ chain have at least one functional domain linked to their C-terminus.

[0010] More preferably, i) the at least one functional domain is linked to the C-terminus of the α chain and / or the β chain directly or via a linker, or ii) the at least one functional domain is linked to the C-terminus of the γ chain and / or the δ chain directly or via a linker.

[0011] Preferably, i) the intracellular regions of the α chain and / or β chain of the synthetic T cell receptor antigen receptor are deleted, or ii) the intracellular regions of the γ chain and / or δ chain of the synthetic T cell receptor antigen receptor are deleted.

[0012] More preferably, i) the functional domain is linked directly or via a linker to the C-terminus of the α chain and / or β chain from which the intracellular region has been deleted, or ii) the functional domain is linked directly or via a linker to the C-terminus of the γ chain and / or δ chain from which the intracellular region has been deleted.

[0013] Preferably, i) the synthetic T cell receptor antigen receptor has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains linked to the C-terminus of the alpha chain and / or the synthetic T cell receptor antigen receptor has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains linked to the C-terminus of the beta chain, or ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the gamma chain of said synthetic T cell receptor antigen receptor and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the delta chain of said STAR.

[0014] In one particular embodiment of the invention, the (two or more) functional domains are linked together directly or via a linker.

[0015] In one specific embodiment of the present invention, at least one functional domain is linked to the C-terminus of the α chain of the synthetic T cell receptor antigen receptor. The intracellular region of the α chain is deleted. The functional domain is linked to the C-terminus of the α chain from which the intracellular region has been deleted via a linker.

[0016] In one specific embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain linked to the C-terminus of a β chain, the intracellular region of which is deleted, and the functional domain linked to the C-terminus of the β chain from which the intracellular region has been deleted via a linker.

[0017] In one specific embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain linked to the C-terminus of a gamma chain, the intracellular region of which is deleted, and the functional domain is linked via a linker to the C-terminus of the gamma chain from which the intracellular region has been deleted.

[0018] In one specific embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain linked to the C-terminus of a δ chain, the intracellular region of which is deleted, and the functional domain is linked via a linker to the C-terminus of the δ chain from which the intracellular region has been deleted.

[0019] Preferably, i) the functional domains linked to the C-terminus of the α chain and / or β chain of said synthetic T cell receptor antigen receptor are identical or different; Alternatively, ii) in said synthetic T cell receptor antigen receptor, the functional domains linked to the C-terminus of the gamma and / or delta chains are the same or different.

[0020] In one particular embodiment of the invention, the multiple functional domains linked to the alpha chain of the synthetic T cell receptor antigen receptor may be the same or different.

[0021] In one particular embodiment of the invention, in said synthetic T cell receptor antigen receptor, the multiple functional domains linked to the β chains may be the same or different.

[0022] In one particular embodiment of the invention, in said synthetic T cell receptor antigen receptor, the functional domains linked to the gamma chain may be the same or different.

[0023] In one particular embodiment of the invention, in said synthetic T cell receptor antigen receptor, the functional domains linked to the δ chain may be the same or different.

[0024] In one particular embodiment of the invention, the functional domain linked to the α chain and the functional domain linked to the β chain of the synthetic T cell receptor antigen receptor may be the same or different.

[0025] In one particular embodiment of the invention, in said synthetic T cell receptor antigen receptor, the functional domain linked to the gamma chain and the functional domain linked to the delta chain may be the same or different.

[0026] Preferably, the functional domain is a costimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. More preferably, the functional domain is an intracellular domain of a costimulatory molecule, an intracellular domain of a co-inhibitory molecule, an intracellular domain of a cytokine receptor, or an intracellular protein, and may be a fusion of a cytokine receptor intracellular domain with a human STAT5 activation module (the amino acid sequence of which is shown in SEQ ID NO: 25) directly or via a linker.

[0027] The costimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137), or CD27.

[0028] The co-inhibitory molecule is selected from TIM3, PD1, CTLA4, and LAG3.

[0029] The cytokine receptor is selected from an interleukin receptor (e.g., IL-2 receptor), an interferon receptor, a tumor necrosis factor superfamily receptor, a colony-stimulating factor receptor, a chemokine receptor, a growth factor receptor, or other membrane protein.

[0030] The intracellular protein is a T cell regulatory factor, such as a NIK domain.

[0031] In one particular embodiment of the invention, the costimulatory molecule is CD40, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:10.

[0032] In one particular embodiment of the invention, the costimulatory molecule is OX40, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:11.

[0033] In one particular embodiment of the invention, the costimulatory molecule is ICOS, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:12.

[0034] In one particular embodiment of the invention, the costimulatory molecule is CD28, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:13.

[0035] In one particular embodiment of the invention, the costimulatory molecule is 4-1BB, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:14.

[0036] In one particular embodiment of the invention, the costimulatory molecule is CD27, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:15.

[0037] In one particular embodiment of the invention, said cytokine receptor is IL-2β, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:22.

[0038] In one particular embodiment of the invention, said cytokine receptor is IL-7α, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:23.

[0039] In one particular embodiment of the invention, said cytokine receptor is IL-21, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:24.

[0040] In one particular embodiment of the invention, said functional domain is a fusion of the IL-2β intracellular domain and the human STAT5 activation module and comprises the amino acid sequence shown in SEQ ID NO:26.

[0041] In one particular embodiment of the invention, said functional domain is a fusion of the IL-7α intracellular domain and the human STAT5 activation module and comprises the amino acid sequence shown in SEQ ID NO:27.

[0042] Preferably, said first constant region is a TCR alpha chain constant region or a TCR gamma chain constant region, preferably a modified TCR alpha chain constant region or a modified TCR gamma chain constant region.

[0043] The constant region of the TCR α chain is selected from the constant region of the human TCR α chain or the constant region of the rodent (preferably murine, more preferably murine) TCR α chain, and the constant region of the TCR γ chain is selected from the constant region of the human TCR γ chain or the constant region of the rodent (preferably murine, more preferably murine) TCR γ chain.

[0044] In one particular embodiment of the invention, the amino acid sequence of the constant region of the human TCR alpha chain is shown in SEQ ID NO:1 and the amino acid sequence of the constant region of the rodent (preferably murine, more preferably mouse) TCR alpha chain is shown in SEQ ID NO:3.

[0045] In one particular embodiment of the present invention, the amino acid sequence of the constant region of the human TCR gamma chain is set forth in SEQ ID NO:45, and the amino acid sequence of the constant region of the rodent (preferably murine, more preferably mouse) TCR gamma chain is set forth in SEQ ID NO:46.

[0046] The constant region of the modified TCR alpha chain is derived from the constant region of a human TCR alpha chain and contains one or more modifications at positions 48, 116 or 119 relative to the constant region of a wild-type human TCR alpha chain, the modifications being mutations or deletions.

[0047] The modified TCR α chain constant region is derived from the human TCR α chain constant region and contains a threonine T to cysteine ​​C mutation at position 48 relative to the wild-type human TCR α chain constant region.

[0048] The constant region of the modified TCR α chain is derived from the constant region of the human TCR α chain and includes a mutation of serine S to leucine L at position 116 and a mutation of glycine G to valine V at position 119 relative to the constant region of the wild-type human TCR α chain.

[0049] The modified TCR α chain constant region is derived from the human TCR α chain constant region and includes, relative to the wild-type human TCR α chain constant region, a threonine T to cysteine ​​C mutation at position 48, a serine S to leucine L mutation at position 116, and a glycine G to valine V mutation at position 119.

[0050] The modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain and contains one or more modifications at positions 6, 13, 15-18, 48, 112, 114, and 115 relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR α chain, wherein the modifications are mutations or deletions.

[0051] The constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises one or more modifications at positions 13, 36, 47, 53, 58, 78, 98, 122 relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain, wherein the modifications are mutations or deletions.

[0052] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes an introduced cysteine ​​relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0053] More preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes a mutation at amino acid position 48, e.g., threonine T to cysteine ​​C, relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0054] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising the introduction of a cysteine ​​comprises the amino acid sequence shown in SEQ ID NO:5.

[0055] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and contains hydrophobic amino acid mutations relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0056] More preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises a change at amino acid position 112, e.g., serine S to leucine L, a change at amino acid position 114, e.g., methionine M to isoleucine I, and / or a change at amino acid position 115, e.g., glycine G to valine V, relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0057] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising hydrophobic amino acid mutations comprises the amino acid sequence shown in SEQ ID NO:7.

[0058] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises an N-terminal modification relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0059] More preferably, the constant region of the modified TCR α chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR α chain, a substitution of the amino acid at position 6, for example E with D, a substitution of K at position 13 with R, and a deletion of amino acids at positions 15 to 18.

[0060] Preferably, the constant region of said modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes a lysine to arginine mutation in the transmembrane region.

[0061] More preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises a substitution of amino acid K at position 122 by R relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0062] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising a lysine to arginine mutation in the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:8.

[0063] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes the introduction of cysteines and hydrophobic amino acid mutations relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0064] More preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises, relative to the wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., threonine T to cysteine ​​C, a change at amino acid position 112, e.g., serine S to leucine L, a change at amino acid position 114, e.g., methionine M to isoleucine I, and a change at amino acid position 115, e.g., glycine G to valine V.

[0065] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising the introduction of cysteines and hydrophobic amino acid mutations comprises the amino acid sequence shown in SEQ ID NO:30.

[0066] The constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain, a mutation at amino acid position 48, e.g., threonine T to cysteine ​​C, and a substitution of amino acid K at position 122 with R.

[0067] The constant region of the modified TCR α chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR α chain, an amino acid at position 6, e.g., a substitution of E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15 to 18, and a mutation of an amino acid at position 48, e.g., threonine T to cysteine ​​C.

[0068] The modified TCR alpha chain constant region is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises, relative to the wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., threonine T, to cysteine ​​C; a change at amino acid position 112, e.g., serine S, to leucine L; a change at amino acid position 114, e.g., methionine M, to isoleucine I; a change at amino acid position 115, e.g., glycine G, to valine V; and a substitution of amino acid K at position 122 with R.

[0069] The modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain and includes, relative to the wild-type rodent (preferably murine, more preferably murine) TCR α chain constant region, a substitution of the amino acid at position 6, e.g., E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15 to 18, a mutation of the amino acid at position 48, e.g., threonine T to cysteine ​​C, a change of the amino acid at position 112, e.g., serine S to leucine L, a change of the amino acid at position 114, e.g., methionine M to isoleucine I, a change of the amino acid at position 115, e.g., glycine G to valine V, and a substitution of the amino acid at position 122, K, with R.

[0070] The constant region of the modified TCR α chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain, and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR α chain, a substitution of the amino acid at position 6, e.g., E with D, a substitution of K at position 13 with R, a deletion of amino acids at positions 15 to 18, a mutation of the amino acid at position 48, e.g., threonine T to cysteine ​​C, and a substitution of the amino acid at position 122 with R.

[0071] The modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain, and includes, relative to the wild-type rodent (preferably murine, more preferably murine) TCR α chain constant region, an amino acid at position 6, e.g., a substitution of E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15 to 18, a change of an amino acid at position 112, e.g., serine S to leucine L, a change of an amino acid at position 114, e.g., methionine M to isoleucine I, and a change of an amino acid at position 115, e.g., glycine G to valine V.

[0072] The constant region of the modified TCR α chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR α chain, a substitution of amino acid at position 6, such as E for D, a substitution of K at position 13 for R, a deletion of amino acids at positions 15 to 18, and a substitution of amino acid K at position 122 for R.

[0073] The modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain, and includes, relative to the wild-type rodent (preferably murine, more preferably murine) TCR α chain constant region, the following changes: an amino acid at position 6, e.g., a substitution of E with D; a substitution of K with R at position 13; a deletion of amino acids at positions 15 to 18; a change of an amino acid at position 112, e.g., serine S, to leucine L; a change of an amino acid at position 114, e.g., methionine M, to isoleucine I; a change of an amino acid at position 115, e.g., glycine G, to valine V; and a substitution of amino acid K with R at position 122.

[0074] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises the introduction of cysteines, hydrophobic amino acid mutations and N-terminal modifications relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR alpha chain.

[0075] More preferably, the constant region of the modified TCR α chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR α chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR α chain, an amino acid at position 6, e.g., a substitution of E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15 to 18, a mutation of an amino acid at position 48, e.g., threonine T to cysteine ​​C, a change of an amino acid at position 112, e.g., serine S to leucine L, a change of an amino acid at position 114, e.g., methionine M to isoleucine I, and a change of an amino acid at position 115, e.g., glycine G to valine V.

[0076] In one specific embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising the introduction of a cysteine, hydrophobic amino acid mutations and deletion of the intracellular region comprises the amino acid sequence shown in SEQ ID NO:16.

[0077] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and includes N-terminal modifications and the introduction of cysteines and hydrophobic amino acid mutations.

[0078] More preferably, the modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain and comprises a substitution of the amino acid at position 6, e.g., E with D, a substitution of K at position 13 with R, a deletion of amino acids at positions 15 to 18, and a mutation of the amino acid at position 48, e.g., threonine T to cysteine ​​C, a change of the amino acid at position 112, e.g., serine S to leucine L, a change of the amino acid at position 114, e.g., methionine M to isoleucine I, and a change of the amino acid at position 115, e.g., glycine G to valine V.

[0079] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising an N-terminal modification and the introduction of a cysteine ​​and a hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO:31.

[0080] Preferably, the constant region of the modified TCR alpha chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR alpha chain and comprises intracellular deletions, N-terminal modifications, and the introduction of cysteines and hydrophobic amino acid mutations.

[0081] More preferably, the modified TCR α chain constant region is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain and comprises an intracellular deletion and a mutation of an amino acid at position 48, e.g., threonine T to cysteine ​​C, an amino acid at position 112, e.g., serine S to leucine L, an amino acid at position 114, e.g., methionine M to isoleucine I, an amino acid at position 115, e.g., glycine G to valine V, and an amino acid at position 6, e.g., substitution of E with D, substitution of K with R at position 13, and deletion of amino acids at positions 15 to 18.

[0082] In one specific embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR α chain comprising intracellular deletions, N-terminal modifications, and the introduction of cysteines and hydrophobic amino acid mutations comprises the amino acid sequence shown in SEQ ID NO:43.

[0083] In one particular embodiment of the invention, said first constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 1, 3, 5, 7, 8, 16, 30, 31 or 43.

[0084] Preferably, said second constant region is a TCR β chain constant region or a TCR δ chain constant region, preferably a modified TCR β chain constant region or a modified TCR δ chain constant region.

[0085] More preferably, the constant region of the TCR β chain is selected from the constant region of a human TCR β chain or the constant region of a rodent (preferably a murine, more preferably a murine) TCR β chain, and the constant region of the TCR δ chain is selected from the constant region of a human TCR δ chain or the constant region of a rodent (preferably a murine, more preferably a murine) TCR δ chain.

[0086] In one particular embodiment of the invention, the constant region of the human TCR β chain comprises the amino acid sequence shown in SEQ ID NO:2.

[0087] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain comprises the amino acid sequence shown in SEQ ID NO:4.

[0088] In one particular embodiment of the invention, the constant region of the human TCR delta chain comprises the amino acid sequence shown in SEQ ID NO:47.

[0089] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR delta chain comprises the amino acid sequence shown in SEQ ID NO:48.

[0090] The constant region of the modified TCR β chain is derived from the constant region of a human TCR β chain and contains one or more modifications at positions 57, 173 or 175 relative to the constant region of a wild-type human TCR β chain, the modifications being mutations or deletions.

[0091] The modified TCR β chain constant region is derived from the human TCR β chain constant region and contains a serine S to cysteine ​​C mutation at position 57 relative to the wild-type human TCR β chain constant region.

[0092] The constant region of the modified TCR β chain is derived from the constant region of the human TCR β chain and contains lysine K to arginine mutations at positions 173 and 175 relative to the constant region of the wild-type human TCR β chain.

[0093] The constant region of the modified TCR β chain is derived from the constant region of the human TCR β chain and includes a mutation of serine S at position 57 to cysteine ​​C and mutations of lysine K at positions 173 and 175 to arginine relative to the constant region of the wild-type human TCR β chain.

[0094] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and contains one or more modifications at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170 relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain, wherein the modifications are mutations or deletions.

[0095] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and comprises one or more modifications at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, 170 relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain, wherein the modifications are mutations or deletions.

[0096] Preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and includes an introduced cysteine ​​relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain.

[0097] More preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain, and relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain, the amino acid at position 56, e.g., serine S, is mutated to cysteine ​​C.

[0098] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain comprising said cysteine ​​introduction comprises the amino acid sequence shown in SEQ ID NO:6.

[0099] Preferably, the constant region of said modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain, with lysines in the intracellular region being replaced by arginines.

[0100] More preferably, the constant region of said modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain, wherein the lysine at position 150, 168 or 170 is replaced by an arginine.

[0101] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain comprising a substitution of lysine with arginine in the intracellular region comprises the amino acid sequence shown in SEQ ID NO:9.

[0102] Preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and comprises an N-terminal modification relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain.

[0103] More preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably a murine, more preferably a mouse) TCR β chain, and relative to the constant region of a wild-type rodent (preferably a murine, more preferably a mouse) TCR β chain, the amino acid at position 3, e.g., R, is substituted with K, the amino acid at position 6, e.g., T, is substituted with F, the K at position 9 is substituted with E, the S at position 11 is substituted with A, the L at position 12 is substituted with V, and the amino acids at positions 17 and 21 to 25 are deleted.

[0104] Preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and includes an introduction of a cysteine ​​and a deletion of the intracellular region relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain.

[0105] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain comprising the introduction of said cysteine ​​and deletion of the intracellular region comprises the amino acid sequence shown in SEQ ID NO:17.

[0106] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and comprises, relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain, a mutation at amino acid position 56, e.g., serine S to cysteine ​​C, and a substitution of lysine at position 150, 168 or 170 with arginine.

[0107] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR β chain and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR β chain, a substitution of an amino acid at position 3, e.g., R with K, an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, and deletion of amino acids at positions 17 and 21 to 25, a mutation of an amino acid at position 56, e.g., serine S to cysteine ​​C, and a substitution of lysine at position 150, 168 or 170 with arginine.

[0108] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR β chain, and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR β chain, a substitution of an amino acid at position 3, e.g., R with K, a substitution of an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, and deletion of amino acids at positions 17 and 21 to 25, and a substitution of lysine at position 150, 168 or 170 with arginine.

[0109] Preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and includes an N-terminal modification and the introduction of a cysteine ​​relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain.

[0110] The constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR β chain, and includes, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR β chain, a substitution of an amino acid at position 3, e.g., R with K, an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, and deletion of amino acids at positions 17 and 21 to 25, and a mutation of an amino acid at position 56, e.g., serine S to cysteine ​​C.

[0111] In one particular embodiment of the invention, the constant region of the rodent (preferably murine, more preferably mouse) TCR β chain comprising the N-terminal modification and introduction of a cysteine ​​comprises the amino acid sequence shown in SEQ ID NO:32.

[0112] Preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain and comprises intracellular deletions, N-terminal modifications, and the introduction of a cysteine ​​relative to the constant region of a wild-type rodent (preferably murine, more preferably mouse) TCR β chain.

[0113] More preferably, the constant region of the modified TCR β chain is derived from the constant region of a rodent (preferably murine, more preferably murine) TCR β chain and comprises, relative to the constant region of a wild-type rodent (preferably murine, more preferably murine) TCR β chain, an intracellular deletion, a mutation of an amino acid at position 56, e.g., serine S to cysteine ​​C, a substitution of an amino acid at position 3, e.g., R with K, a substitution of an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, and deletions of amino acids at positions 17 and 21-25.

[0114] In one particular embodiment of the invention, the constant region of a rodent (preferably murine, more preferably mouse) TCR β chain comprising the intracellular deletion, N-terminal modification, and introduction of a cysteine ​​comprises the amino acid sequence shown in SEQ ID NO:44.

[0115] In one particular embodiment of the invention, said second constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 2, 4, 6, 9, 17, 32 or 44.

[0116] The target binding region is located N-terminal to the constant region, and the two may be directly linked or linked via a linker.

[0117] The first target binding region may comprise one or more identical or different binding regions, which may be an antigen binding region or fragment thereof, a non-immunocyte protein antigen binding domain or fragment thereof, an antibody binding region or fragment thereof, a receptor or fragment thereof, or a ligand or fragment thereof, preferably a natural T cell receptor.

[0118] The antigen-binding region is derived from an antibody.

[0119] said STARs comprise one or more antigen binding regions, Preferably, the antigen-binding regions are the same or different; More preferably, the multiple antigen-binding regions are linked directly or via a linker.

[0120] The antibody may be a monoclonal or polyclonal antibody.

[0121] The antibody also ab , F ab ', F ab '-SH, Fv, scFv, (F ab ')2, may be a fragment including a single domain antibody, a diabody (dAb) or a linear antibody.

[0122] The antibody may be a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody).

[0123] Preferably, the antibody may be a fully human antibody, a humanized antibody, or an antibody derived from an animal, such as a mouse, rabbit, cow, or monkey.

[0124] Preferably, the first target binding region is linked to the first constant region either directly or via a linker, and / or the second target binding region is linked to the second constant region either directly or via a linker.

[0125] Preferably, the first target binding region and the second target binding region each independently or in combination specifically bind to a target antigen.

[0126] Preferably, the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, such as LILRB4, GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, mammary ductal epithelial mucoprotein, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, α alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA. IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mutated hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, active enzyme and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor I (IGF1)-I, IGF-II, IGF-I receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, additional domain A (EDA) and additional domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC) A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endothelin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), or FCRL5 (UNIPROTIt is a cancer-associated antigen selected from the group consisting of:

[0127] Preferably, the first target binding region comprises one or more antibodies or antibody fragments of the specific binding target antigen, and the second target binding region comprises one or more antibodies or antibody fragments of the specific binding target antigen.

[0128] The antibodies or antibody fragments are linked together either directly or via a linker.

[0129] Preferably, the first target binding region comprises one or more single chain antibodies or one or more single domain antibodies that specifically bind to the target antigen, and / or the second target binding region comprises one or more single chain antibodies or one or more single domain antibodies that specifically bind to the target antigen.

[0130] The single chain antibodies are linked together either directly or via a linker. The single domain antibodies are linked together either directly or via a linker.

[0131] Preferably, the single chain antibody comprises a heavy chain variable region and a light chain variable region that are linked directly or via a linker.

[0132] Preferably, the multiple antigen-binding regions in the first target-binding region and / or the second target-binding region bind the same or different target antigens.

[0133] Preferably, the multiple antigen-binding regions in the first target-binding region and / or the second target-binding region bind different regions, e.g., different epitopes, of the same target antigen.

[0134] In one particular embodiment of the invention, the target antigen is LILRB4.

[0135] In one particular embodiment of the invention, the antigen-binding region of the first target binding region comprises one or more single domain antibodies, and / or the antigen-binding region of the second target binding region comprises one or more single domain antibodies; Preferably, the single domain antibodies contained in the antigen-binding region of the first target-binding region are identical or different, Preferably, the single domain antibodies comprised in the antigen-binding region of said second target-binding region are identical or different, More preferably, the single domain antibodies are linked directly or via a linker.

[0136] Preferably, the single domain antibody comprised in the antigen-binding region of said first target binding region and the single domain antibody comprised in the antigen-binding region of said second target binding region are the same or different.

[0137] In one specific embodiment of the present invention, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDRs 1 to 3, i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35; or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO:36, CDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and said CDR3 comprises the amino acid sequence shown in SEQ ID NO:38.

[0138] In one particular embodiment of the invention, said single domain antibody comprises the amino acid sequence shown in SEQ ID NO:28 or 29.

[0139] In a second aspect of the invention there is provided a STAR complex comprising: i) the STAR complex comprises an α chain, a β chain, CD3ε, CD3γ, CD3δ, and CD3ζ, and the α chain comprises a first target binding region and a first constant region, and the β chain comprises a second target binding region and a second constant region, or the α chain comprises a first target binding region and the β chain comprises a second target binding region and a second constant region, or ii) the STAR complex comprises a gamma chain, a delta chain, CD3epsilon, CD3gamma, CD3delta, and CD3zeta, wherein the gamma chain comprises a first target binding region and a first constant region and the delta chain comprises a second target binding region and a second constant region, or wherein the gamma chain comprises a first target binding region and a first constant region and the delta chain comprises a second target binding region and a second constant region, providing a STAR complex.

[0140] Preferably, i) at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ, and CD3ζ has at least one functional domain linked to its C-terminus, or ii) at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ has at least one functional domain linked to its C-terminus.

[0141] Preferably, i) the at least one functional domain is linked directly or via an anchor to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ, and CD3ζ, or ii) the at least one functional domain is linked directly or via an anchor to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ.

[0142] Preferably, i) the intracellular region of at least one of the alpha chain, beta chain, CD3ε, CD3γ, CD3δ, and CD3ζ of the STAR complex is deleted, or ii) the intracellular region of at least one of the gamma chain, delta chain, CD3ε, CD3γ, CD3δ, and CD3ζ of the STAR complex is deleted.

[0143] More preferably, i) the at least one functional domain is linked directly or via an anchor to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ, and CD3ζ from which the intracellular region has been deleted, or ii) the at least one functional domain is linked directly or via an anchor to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ from which the intracellular region has been deleted.

[0144] In one specific embodiment of the invention, i) at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ, and CD3ζ of the STAR complex has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains linked to the C-terminus, or ii) at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ of the STAR complex has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains linked to the C-terminus,

[0145] Preferably, the functional domains are directly linked to each other, or may be linked via a linker.

[0146] Preferably, i) the functional domains linked to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ, and CD3ζ of said STAR complex are identical or different, or ii) the functional domains linked to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ of said STAR complex are identical or different.

[0147] In one specific embodiment of the present invention, the functional domains linked to the α chain may be the same or different. The functional domains linked to the β chain may be the same or different. The functional domains linked to the γ chain may be the same or different. The functional domains linked to the δ chain may be the same or different. The functional domains linked to the CD3ε may be the same or different. The functional domains linked to the CD3γ may be the same or different. The functional domains linked to the CD3δ may be the same or different. The functional domains linked to the CD3ζ may be the same or different.

[0148] In one particular embodiment of the invention, the functional domains linked to said α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ, respectively, may be identical or different.

[0149] In one particular embodiment of the invention, the functional domains linked to said gamma chain, delta chain, CD3ε, CD3γ, CD3δ, and CD3ζ, respectively, may be identical or different.

[0150] Preferably, the functional domain is a costimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. More preferably, the functional domain is an intracellular domain of a costimulatory molecule, an intracellular domain of a co-inhibitory molecule, an intracellular domain of a cytokine receptor, or an intracellular protein. Preferably, the costimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137) or CD27; Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, LAG3; Preferably, the cytokine receptor is selected from an interleukin receptor (e.g., IL-2 receptor), an interferon receptor, a tumor necrosis factor superfamily receptor, a colony-stimulating factor receptor, a chemokine receptor, a growth factor receptor, or other membrane protein; Preferably, the intracellular protein is a T cell regulatory factor, such as a NIK domain.

[0151] In one particular embodiment of the invention, the costimulatory molecule is CD40, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:10.

[0152] In one particular embodiment of the invention, the costimulatory molecule is OX40, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:11.

[0153] In one particular embodiment of the invention, the costimulatory molecule is ICOS, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:12.

[0154] In one particular embodiment of the invention, the costimulatory molecule is CD28, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:13.

[0155] In one particular embodiment of the invention, the costimulatory molecule is 4-1BB, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:14.

[0156] In one particular embodiment of the invention, the costimulatory molecule is CD27, the intracellular domain of which comprises the amino acid sequence shown in SEQ ID NO:15.

[0157] In one particular embodiment of the invention, said STAR complex comprises a STAR as defined above, as well as CD3ε, CD3γ, CD3δ, and CD3ζ.

[0158] Preferably, said CD3ε, CD3γ, CD3δ and / or CD3ζ are derived from humans.

[0159] In one particular embodiment of the invention, said CD3ε comprises the amino acid sequence shown in SEQ ID NO:20.

[0160] In one particular embodiment of the invention, said CD3γ comprises the amino acid sequence shown in SEQ ID NO:18.

[0161] In one particular embodiment of the invention, said CD3δ comprises the amino acid sequence shown in SEQ ID NO:19.

[0162] In one particular embodiment of the invention, said CD3ζ comprises the amino acid sequence shown in SEQ ID NO:21.

[0163] In a third aspect of the invention, there is provided an antibody or antigen-binding fragment comprising a heavy chain variable region and / or a light chain variable region.

[0164] the heavy chain variable region comprises CDRs 1 to 3, i) CDR1 comprises the amino acid sequence shown in SEQ ID NO:33, CDR2 comprises the amino acid sequence shown in SEQ ID NO:34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:35.

[0165] or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO:36, CDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and said CDR3 comprises the amino acid sequence shown in SEQ ID NO:38.

[0166] The antibody also ab , F ab ', F ab '-SH, Fv, scFv, (F ab')2, may include fragments of single domain antibodies, diabodies (dAbs) or linear antibodies.

[0167] The antibody may be a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody).

[0168] Preferably, the antibody may be a fully human antibody, a humanized antibody, or an antibody derived from an animal, such as a mouse, rabbit, cow, or monkey.

[0169] In one particular embodiment of the invention, the antibody or antigen-binding fragment is a single chain antibody or a single domain antibody.

[0170] In one particular embodiment of the invention, the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:28 or 29.

[0171] In a fourth aspect of the invention, there is provided a single chain antibody comprising a heavy chain variable region and / or a light chain variable region.

[0172] the heavy chain variable region comprises CDRs 1 to 3, i) CDR1 comprises the amino acid sequence shown in SEQ ID NO:33, CDR2 comprises the amino acid sequence shown in SEQ ID NO:34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:35.

[0173] or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO:36, CDR2 comprises the amino acid sequence shown in SEQ ID NO:37, and said CDR3 comprises the amino acid sequence shown in SEQ ID NO:38.

[0174] In one particular embodiment of the invention, the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:28 or 29.

[0175] In a fifth aspect, the present invention provides a single domain antibody comprising a heavy chain variable region comprising CDRs 1 to 3.

[0176] i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO:33, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:34, and said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35, or ii) CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:37, and said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:38.

[0177] In one particular embodiment of the invention, said single domain antibody comprises the amino acid sequence shown in SEQ ID NO:28 or 29.

[0178] In a sixth aspect, the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, or the above-mentioned single domain antibody, which method comprises preparing a phage display library and screening the antibody or antigen-binding fragment or single domain antibody from the phage display library.

[0179] In a seventh aspect of the present invention, there is provided an antigen receptor comprising a transmembrane region, an intracellular region and one or more identical or different extracellular binding domains.

[0180] The antigen receptor is a TCR or a CAR.

[0181] In one particular embodiment of the invention, said antigen receptor is a CAR.

[0182] The extracellular binding domain is an extracellular antigen binding domain, an extracellular antibody binding domain, a receptor, or a ligand, and the receptor is preferably a natural T cell receptor.

[0183] In one particular embodiment of the invention, said extracellular binding domain is an extracellular antigen binding domain.

[0184] The extracellular antigen-binding domain is derived from an autoantibody.

[0185] Preferably, the transmembrane region and one or more extracellular antigen-binding domains are directly linked or linked via a linker.

[0186] Preferably, the antigen is a cancer-associated antigen, such as LILRB4, GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, mammary ductal epithelial mucoprotein, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, α-alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA. IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mutated hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, active enzyme and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor I (IGF1)-I, IGF-II, IGF-I receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, additional domain A (EDA) and additional domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC) A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endothelin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), or FCRL5 (UNIPROTIt is a cancer-associated antigen selected from the group consisting of:

[0187] Preferably, the antigen is LILRB4.

[0188] Preferably, the extracellular antigen-binding domain comprises CDR1 to CDR3, wherein i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35. Alternatively, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0189] In one particular embodiment of the invention, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment as described above, a single-chain antibody as described above, or a single-domain antibody as described above.

[0190] Preferably, the transmembrane region is derived from human CD8.

[0191] Preferably, the intracellular region is derived from 4-1BB, CD28 or CD3ζ.

[0192] In an eighth aspect of the invention there is provided a nucleic acid encoding said STAR, said STAR complex, said antibody or antigen-binding fragment, said single chain antibody, said single domain antibody, said antigen receptor.

[0193] In a ninth aspect of the present invention, there is provided a vector comprising the above nucleic acid.

[0194] The vector can be expressed in vivo, in vitro, or ex vivo, and is preferably an expression vector, such as a prokaryotic expression vector, a viral expression vector, a plasmid, a cosmid, a phage, or a virus.

[0195] Preferably, the prokaryotic expression vector is an E. coli series, such as pET-26b or pET28a+.

[0196] Preferably, the expression vector is Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloproliferative virus 29 (MC29), or avian myeloblastosis virus (AEV). More preferably, the expression vector is a lentiviral expression vector, such as pHAGE-IRES-RFP.

[0197] In a tenth aspect of the present invention, there is provided a host cell comprising the above nucleic acid or the above vector.

[0198] Preferably, the host cell may be a eukaryotic cell or a prokaryotic cell, and more preferably, the host cell is a yeast cell, a 293 cell, a CHO cell, an E. coli cell, or the like.

[0199] In an eleventh aspect of the invention there is provided an immune cell expressing the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen receptor.

[0200] Preferably, said immune cells contain one or more of the above nucleic acids.

[0201] Preferably, said immune cells are selected from T cells, Treg cells, macrophages, NK cells, NKT cells, peripheral blood mononuclear cells, TIL cells or dendritic cells (DCs).

[0202] Preferably, the immune cells are isolated from the subject's T cells.

[0203] In one particular embodiment of the invention, said immune cells are selected from T cells, NK cells, CTLs, human embryonic stem cells, lymphoid progenitor cells and / or T cell progenitor cells.

[0204] In a twelfth aspect of the present invention, there is provided a CAR-T cell comprising the above-mentioned antibody or antigen fragment, the above-mentioned single chain antibody or the above-mentioned single domain antibody.

[0205] In a thirteenth aspect of the present invention, there is provided a method for preparing immune cells obtained by transmitting the above-mentioned nucleic acid sequence to immune cells and expressing the same.

[0206] In a fourteenth aspect of the present invention, Step 1) of obtaining the nucleic acid from the positive T cells; Step 2) isolating and culturing primary T cells; and step 3) delivering the nucleic acid obtained in step 1) to the primary T cell described in step 2) to obtain a recombinant T cell expressing said STAR.

[0207] In a fifteenth aspect of the present invention, (1) a step of obtaining the nucleic acid from positive T cells by screening; Step (2) of ligating the nucleic acid obtained in step (1) into a vector backbone to obtain an expression vector; Step (3) of transforming the expression vector obtained in step (2) into a host cell and then inducing expression thereof; (4) obtaining a STAR.

[0208] In a sixteenth aspect of the present invention there is provided a method for preparing an antibody or antigen-binding fragment, single chain antibody or single domain antibody, comprising Western blotting and / or DNA immunization.

[0209] In a seventeenth aspect of the present invention, A) obtaining the encoded nucleic acid sequence; and (B) transforming the nucleic acid sequence obtained in step A) into a host cell, followed by inducing expression and purifying the antibody or antigen-binding fragment, single-chain antibody or single-domain antibody.

[0210] In an eighteenth aspect of the invention there is provided the use of a STAR as defined above, a STAR complex as defined above, an antibody or antigen-binding fragment as defined above, a single domain antibody as defined above, an antigen receptor as defined above, a nucleic acid as defined above, an immune cell as defined above in the preparation of a product for diagnosing or treating a tumour.

[0211] Preferably, the tumor comprises lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, malignant melanoma, myelodysplastic syndrome, and sarcoma.

[0212] Preferably, said leukemia comprises acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; Preferably, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; Preferably, the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma; Preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia; Preferably, the chronic myelogenous leukemia is chronic myelomonocytic leukemia.

[0213] In a nineteenth aspect of the present invention, there is provided an immunoconjugate or antibody-drug complex comprising an above-mentioned antibody or antigen-binding fragment, above-mentioned single-chain antibody or above-mentioned single-domain antibody of the invention conjugated to a therapeutic or diagnostic reagent.

[0214] In a 20th aspect of the invention there is provided a pharmaceutical composition comprising said STAR, said STAR complex, said antibody or antigen-binding fragment, said single domain antibody, said antigen receptor, said nucleic acid, said immune cell.

[0215] Preferably, the drug further comprises a pharmaceutically acceptable adjuvant, more preferably, the pharmaceutically acceptable adjuvant includes, but is not limited to, a diluent, a binder, a wetting agent, a surfactant, a lubricant, or a disintegrant.

[0216] In a twenty-first aspect of the invention there is provided a kit comprising the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen receptor, the above-mentioned nucleic acid, the above-mentioned immune cell.

[0217] In a twenty-second aspect of the invention there is provided a method for treating a tumour comprising administering to a subject an effective amount of a STAR, STAR complex, CAR, antibody or antigen-binding fragment thereof, single chain antibody, single domain antibody, immune cell, CAR-T cell or pharmaceutical composition according to the invention.

[0218] The "linker" according to the present invention includes, but is not limited to, a rigid linker, a flexible linker, a cleavable linker, or a nonsense amino acid. Preferably, the amino acid sequence of the rigid linker is one or more selected from SEQ ID NOs: 49 to 59. Preferably, the flexible linker is selected from glycine and / or serine-rich peptide fragments, preferably, the flexible linker is one or more selected from SEQ ID NOs: 60 to 112, and preferably, the cleavable linker is one or more selected from SEQ ID NOs: 113 to 117.

[0219] An "antibody" according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2).

[0220] "Antigen-binding fragments" according to the present invention include Fab fragments comprising the VL, CL, VH, and CH1 domains, Fab' fragments which are Fab fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain, Fd fragments comprising the VH and CH1 domains, Fd' fragments comprising the VH, CH1 domains, and one or more cysteine ​​residues at the C-terminus of the CH1 domain, Fv fragments comprising the VL and VH domains of a single arm of an antibody, dAb fragments consisting of the VH or VL domain, isolated CDR regions, and a disulfide bridge at the hinge region. Examples of antibodies include, but are not limited to, F(ab')2 fragments, which are bivalent fragments comprising two Fab' fragments linked by a single chain; single-chain antibody molecules (e.g., single-chain Fv; scFv); "diabodies" having two antigen-binding sites comprising a heavy-chain variable region (VH) linked to a light-chain variable region (VL) of the same polypeptide chain; "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with a complementary light-chain polypeptide, form a pair of antigen-binding regions; and any modified forms of the foregoing that retain antigen-binding activity.

[0221] "CDR" as used herein refers to the complementarity-determining regions within the variable sequences of an antibody. Each variable region contains three CDRs, designated CDR1, CDR2, and CDR3, in each of the heavy and light chain variable regions. The exact boundaries of these CDRs are defined differently in different systems. The system described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987), and (1991)) not only provides an unambiguous residue numbering system suitable for antibody variable regions, but also provides residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each CDR may comprise amino acid residues from the "complementarity determining regions" defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987), and Chothia et al., Nature 342:877-883 (1989)) have proposed a system for numbering CDRs based on Kabat CDRs. We have discovered that certain subportions within CDRs adopt nearly identical peptide backbone conformations despite significant diversity at the amino acid sequence level. These subportions are designated L1, L2, L3, or H1, H2, and H3, respectively, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions may also be referred to as Chothia CDRs, whose boundaries overlap with those of Kabat CDRs. While other CDR boundary definitions overlap with those of Kabat CDRs even if they do not strictly adhere to either of the above systems, and the methods used herein can utilize CDRs defined according to any of these systems, preferred embodiments utilize CDRs defined by Kabat or Chothia. The term "antibody variable region" refers to the amino acid sequences comprising the complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and framework regions (FR) of the light and heavy chains of an antibody molecule. VH refers to the heavy chain variable domain. VL refers to the light chain variable domain.

[0222] "Diagnosis" according to the present invention refers to determining whether a patient has previously suffered from a disease or condition, is suffering from a disease or condition at the time of diagnosis, or will suffer from a disease or condition in the future, determining the progression or likelihood of future progression of a disease, or assessing a patient's response to treatment.

[0223] As used herein, "treatment" means slowing, interrupting, preventing, controlling, halting, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily mean the complete elimination of all disease-related signs, symptoms, condition, or disorder, and refers to a therapeutic intervention that improves the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to progress.

[0224] An "effective amount" according to the present invention refers to the amount or dose of a STAR, STAR complex, CAR, CAR-T, STAR-T, immune cell, pharmaceutical composition, etc. according to the present invention that will achieve the desired therapeutic or prophylactic effect when administered once or multiple times to a patient or organ.

[0225] The "product" according to the present invention may be a kit, a chip, an antibody conjugate, a multifunctional antibody, a pharmaceutical composition, etc.

[0226] An "individual" or "subject" according to the present invention may be a human or a non-human animal, and said non-human animal may be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0227] The term "and / or" as used herein includes all combinations of the items connected by that term, and each combination should be considered as if it were individually listed herein. For example, "A and / or B" includes "A," "A and B," and "B." As another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A, B, and C."

[0228] The terms "comprise" and "comprise" used herein are open terms, and when used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends and still have the activity described in the present invention. Furthermore, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a particular expression system), but this does not substantially affect the function of the polypeptide. Therefore, when a specific polypeptide amino acid sequence is described in the specification and claims of this application, the sequence may not contain the methionine encoded by the start codon at the N-terminus, but sequences containing a methionine are also covered, and therefore, the encoding nucleotide sequence may also contain a start codon, and vice versa. [Brief explanation of the drawings]

[0229] The present invention will now be described in detail with reference to the accompanying drawings, in which: FIG. [Figure 1A] Detected results of LILRB4 expression levels in various AML cell lines. [Figure 1B] Detected results of LILRB4 expression levels in various AML cell lines. [Figure 2] Schematic of the structure of a STAR targeting LILRB4. [Figure 3] The killing effect of various antibodies on target cells. [Figure 4A] Affinity results of NLB4 / NLB14 nanoantibodies detected by SPR. [Figure 4B] Affinity results of NLB4 / NLB14 nanoantibodies detected by SPR. [Figure 5A] Competitive binding results of NLB4 / NLB14 nanoantibodies detected by SPR. [Figure 5B]Competitive binding results of NLB4 / NLB14 nanoantibodies detected by SPR. [Figure 6] FIG. 1 is a schematic diagram of the structure of the LILRB4 protein. [Figure 7] This is the epitope recognized by the NLB4 / NLB14 nanobody on LILRB4. [Figure 8] FIG. 1 shows flow cytometry analysis of whether NLB4 / NLB14 nanobodies bind non-specifically to multiple proteins of the LILRB4 family. [Figure 9A] FIG. 1 shows flow cytometry analysis of whether NLB4 / NLB14 nanobodies bind non-specifically to various human tissues. [Figure 9B] FIG. 1 shows flow cytometry analysis of whether NLB4 / NLB14 nanobodies bind non-specifically to various human tissues. [Figure 10] FIG. 1 shows flow cytometry analysis of binding of NLB4 / NLB14 nanoantibodies to human LILRB4 and mouse LILRB4. [Figure 11] Schematic diagram of the structures of single-epitope and dual-epitope STARs targeting LILRB4. [Figure 12] FIG. 1 shows flow cytometry analysis to confirm membrane expression of single-epitope and dual-epitope LILRB4 STAR. [Figure 13] 1 shows the recognition and killing effect of LILRB4 STAR-T cells on the target cell line THP1. [Figure 14A] This is the killing effect of LILRB4 STAR-T cells on target cells. [Figure 14B] This is the killing effect of LILRB4 STAR-T cells on target cells. [Figure 15] Cytokine secretion levels of LILRB4 STAR-T. [Figure 16A] Killing effect of LILRB4 STAR-T on mouse tumor models. [Figure 16B]Killing effect of LILRB4 STAR-T on mouse tumor models. [Figure 16C] Killing effect of LILRB4 STAR-T on mouse tumor models. [Figure 16D] Killing effect of LILRB4 STAR-T on mouse tumor models. [Figure 17] HE staining results of various tissues of mice after reinjection of LILRB4 STAR-T. [Figure 18] 1 shows the results of detecting the in vitro tumorigenicity of LILRB4 STAR-T cells. [Figure 19A] The relationship between NLB4 / NLB14 and the LILRB4 ligand ApoE. [Figure 19B] The relationship between NLB4 / NLB14 and the LILRB4 ligand ApoE. [Figure 19C] The relationship between NLB4 / NLB14 and the LILRB4 ligand ApoE. DETAILED DESCRIPTION OF THE INVENTION

[0230] Hereinafter, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. All other embodiments that a person skilled in the art can obtain without creative work based on the embodiments of the present invention belong to the protection scope of the present invention.

[0231] Experimental Materials and Methods

[0232] Vector construction

[0233] The lentiviral vectors and lentiviral packaging plasmids used in the examples of this application were either purchased from commercial companies or synthesized by commercial companies. Gene fragments used in the examples of this application, including signal peptides, antibody binding regions, hinge regions, TCR constant regions, tag proteins, etc., were all synthesized by commercial companies. One or more target fragments were ligated by synthetic primer PCR to obtain the corresponding functional sequences. The lentiviral vector used in this application is pHAGE-hEF1α-RFP, and the pHAGE-hEF1A-WPRE-AMP vector was obtained using the restriction enzymes SpeI / SalI. Gene fragments were obtained by synthesis and PCR. The complete vector was obtained by homologous recombination under the action of recombinase.

[0234] The α-chain constant region is derived from mouse, and the wild-type is designated TRAC. The β-chain constant region is derived from mouse, and the wild-type is designated TRBC. The costimulatory molecule is OX40. Lentiviral packaging

[0235] 5 × 10 Lentix-293T cells were cultured in a 10 cm culture dish. 5 The cells were inoculated at 100 cells / mL and cultured in a 37°C, 5% CO2 incubator. Transfection was performed when the cell density reached approximately 80% (as observed under a microscope). The four plasmids were homogenously mixed with 500 μL of serum-free DMEM at a ratio of PMD2.G:REV:PMDLG:transfer plasmid = 1:1:2:4. 54 μL of PEI-max was homogenously mixed with 500 μL of serum-free DMEM and left at room temperature for 5 minutes (a volume-to-mass ratio of PEI-max to plasmid was 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently pipetted, mixed homogenously, and left at room temperature for 15 minutes. The final mixture was slowly added to the medium, mixed thoroughly, and then returned to the incubator for an additional 12–16 hours. The medium was then replaced with 6% FBS DMEM and further cultured. Virus solutions were collected after 48 and 72 hours. T cell culture and infection 1)Culture method of Jurkat T cell line

[0236] Jurkat T cell line was cultured in RPMI 1640 medium containing 10% FBS at a density of 3 x 10 5 / ml, maximum 3*10 6 The density was adjusted to 1 / ml or less, and the cells were subcultured every 1-2 days. After counting the number of cells, the required amount of cells was collected, and the medium was replenished to adjust the density to the above-mentioned level, and the cells were cultured in a CO2 incubator. 2) Infection method of Jurkat T cell line

[0237] Count the cells and count 1*10 6 The cells were centrifuged, the medium was replaced, and the cells were resuspended in 1 ml of RPMI 1640 medium containing 10% FBS. The cells were added to a 24-well plate, and the virus solution (MOI = 1) was added. The plate was centrifuged at 1500 rpm for 90 minutes and cultured in a CO2 incubator. 12 hours after infection, the medium was completely replaced with fresh RPMI 1640 medium containing 10% FBS, and the positive rate was detected 72 hours later. 3) Method for culturing human primary T cells

[0238] After obtaining primary T cells by Ficoll separation, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / ml IL-2 at an initial culture density of 1 x 10 6 / ml and added to well plates pre-coated with CD3 and RetroNectin (all at a final concentration of 5 μg / ml). 5 / ml, at most 3 × 10 6 / ml or less and subcultured every 1 to 2 days. 4) Method for infecting human primary T cells

[0239] After culturing primary T cells for 48 hours, virus solution was added, and the cells were centrifuged at 1500 rpm for 90 minutes at an MOI of 20 and placed in a CO2 incubator for culture. 24 hours after infection, the cells were supplemented with X-VIVO medium containing 10% FBS and 100 IU / ml IL-2 and transferred to another well. 72 hours later, infection efficiency was detected using labeled proteins or antibodies. 5) Method for detecting infection efficiency

[0240] 72 hours after infection, cells were homogenously pipetted and counted. 5 The cells were collected at 1000 x g / ml, centrifuged, the supernatant was discarded, and the staining solution was PBS + 2% FBS + 2mM EDTA. The corresponding antibody was added and incubated for 30 minutes, then washed twice with PBS, and loaded onto the device for detection. Detection of in vitro function of STAR-T cells

[0241] Positive T cells were cocultured with luciferase-expressing target cells (Raji cells, CD19-KO Raji cells, or CD22-KO Raji cells) at the indicated effector-target ratios.

[0242] After 24 hours of co-culture, the cell suspension was gently pipetted uniformly. 150 μL of cell suspension was taken from each well and added to a white 96-well plate. Two replicate wells per well were added with luciferase substrate. After 10 minutes of incubation with slow shaking, chemiluminescence readings were detected using a multi-function microplate reader. Cell killing was calculated as follows: Killing efficiency = 100% - (effector cell-target cell well value / control cell-target cell well value). Alternatively, after 24 hours of co-culture, cell culture supernatants were collected and cytokine content was detected using commercially available kits. Detection of in vivo function of STAR-T cells

[0243] We constructed this model using NSG immunodeficient mice. The genotype of these mice is NOD-Prkdcem26Il2rgem26 / Nju, which lacks T cells, B cells, and NK cells, and also has defects in macrophages and dendritic cells. For this experiment, we used female NSG mice aged 6–8 weeks, and controlled the weight difference between mice in each experimental batch to within 2 g. To prevent pathogen contamination, the mice were housed in individual, specific pathogen-free (SPF) ventilated cages and provided with a normal diet and drinking water with a slightly acidic pH.

[0244] A tumor model was established by xenografting Raji cells, a human Burkitt's lymphoma cell line. Raji cells express the luciferase gene via a lentiviral vector. The progression and changes of Raji tumors were monitored in real time in vivo by fluorescein chemiluminescence and bioimaging. In this model, 1–3 × 10 6 Raji luciferase cells were inoculated into 6-8 week-old female NSG mice via tail vein reinjection. Fluorescein potassium salt solution was injected intraperitoneally into the mice, and the fluorescent signal of tumor cells in vivo was detected by intravital imaging. Example 1 Construction of target cells

[0245] To screen for nanobodies targeting LILRB4 and detect the killing effect of STAR-T on AML tumor cells, we detected the expression of LILRB4 antigen in various AML cells by flow cytometry. As shown in Figure 1A and Figure 1B, LILRB4 antigen expression was high in THP1, MV4-11, and OCI-AML3 cells but low in protoplast-formed KASUMI-1 cells. Example 2 Screening of nanobodies targeting LILRB4

[0246] 1. Immunization of alpacas with human LILRB4 protein Healthy alpacas were immunized with the commercially available extracellular domain of human LILRB4 protein (100 μg) using adjuvants including complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified extracellular domain of human LILRB4 protein was diluted in PBS and mixed with the corresponding adjuvant at a 1:1 ratio. The antigen and adjuvant were thoroughly mixed to form a stable emulsion. The antigen mixture was drawn up into a syringe and injected subcutaneously at 100–200 μL aliquots at multiple sites under the skin of the alpaca's neck. The specific animal immunization process was as follows:

[0247] 1) Day 1 (primary immunization): Alpacas were immunized by subcutaneous injection with LILRB4 antigen (100 μg) mixed with complete Freund's adjuvant (CFA).

[0248] 2) Day 14 (second immunization): Alpacas were immunized by subcutaneous injection with LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA).

[0249] 3) Day 28 (third immunization): The alpaca was again immunized by subcutaneous injection with LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA).

[0250] 4) Day 42 (first serum collection): Blood was collected from the alpaca's ear vein, serum was extracted, and antibody titers were detected. The P / N value of the 200,000-fold diluted serum was greater than 2.

[0251] 5) Day 42 (4th immunization): The alpaca was immunized again by subcutaneous injection of LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) to enhance the immune effect.

[0252] 6) Day 53 (second serum collection): Blood samples were taken from the alpaca's ear vein, serum was extracted, and antibody titers were detected. The P / N value of the 200,000-fold diluted serum was greater than 2.

[0253] 7) Days 54, 57, and 60: 30–40 mL of alpaca blood samples were collected from the hind leg vein of the alpaca and PBMCs were isolated. 2.PBMC separation

[0254] 1) PBMCs were isolated in a biosafety cabinet, and the blood in the anticoagulant tube was transferred to a 50 mL centrifuge tube (30 mL). PBS was added to bring the total volume to 50 mL, and the mixture was gently mixed evenly.

[0255] 2) Prepare new 50 mL centrifuge tubes and add 15 mL of Ficoll separation solution per tube. 25 mL of blood sample was layered on top of the Ficoll solution. Stable manipulation was used to form a layer of Ficoll and blood, taking care not to mix the Ficoll and blood.

[0256] 3) The centrifuge was adjusted to a speed of 0, room temperature, and a centrifugal force of 800 g for 30 minutes. After centrifugation, the sample was removed from the centrifuge and separated into an upper aqueous phase, buffy coat layer, Ficoll layer, and red blood cell layer. PBMCs were contained in the buffy coat layer. The buffy coat layer was aspirated and transferred to a new 50 mL centrifuge tube.

[0257] 4) 50 mL of PBS was added to the sample tube and mixed uniformly, followed by centrifugation at 2000 rpm at room temperature for 5 minutes. The supernatant was discarded, and 5 mL of PBS was added to resuspend the cell pellet.

[0258] 5) Repeat step 4 and lower the temperature to 4°C.

[0259] 6) 5 mL of PBS was added to resuspend the cell pellet, and 40 mL of PBS was added to count the number of cells.

[0260] 7) The mixture was centrifuged at 1500 rpm at 4°C for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS and pipetted thoroughly. 20 mL of Trizol was added, mixed thoroughly, and left to stand at room temperature for 5 minutes to lyse the cells. The cells were then dispensed in 1 mL aliquots into RNase-free 1.5 mL EP tubes and frozen at -80°C for RNA extraction. 3. RNA Extraction and Reverse Transcription

[0261] 1) The sample was removed from -80°C, thawed at room temperature, and then 200 μL of chloroform was added and allowed to stand at room temperature for 3 minutes. The sample was centrifuged at 12,000 g for 15 minutes at 4°C to separate the upper aqueous phase, the middle layer, and the organic layer. The upper layer was transferred to a new RNase-free tube, and 1 μL of glycogen and 500 μL of isopropanol were added. The mixture was allowed to stand at 4°C overnight.

[0262] 2) The samples were centrifuged at 12,000 g for 20 minutes at 4°C, the supernatant was removed by aspiration, and the precipitate was washed with 1 mL of pre-chilled 75% alcohol. The precipitate was then centrifuged again to remove the alcohol and air-dried. 15 μL of RNase-free water was added to each tube to dissolve the RNA precipitate, and reverse transcription was performed.

[0263] 3) cDNA was synthesized using a Promega reverse transcription kit (20 μL system).

[0264] Step 1: A fixed amount of template RNA was taken and Oligo(dT) was added (see Table 1).

[0265] [Table 1] Step 2: The mixture of template RNA and Oligo(dT) was pre-denatured at 65°C for 5 minutes and then placed back on ice.

[0266] Step 3: Before pre-denaturation, RT-Mix can be prepared in advance, 8 µL per tube. The ingredients and volumes are listed in Table 2.

[0267] [Table 2] Step 4: Set up the reverse transcription program, extension, and reverse transcriptase inactivation. When the process is complete, cDNA is obtained. 4. Phage Library Construction

[0268] 1) Obtaining VHH sequences by PCR The VHH sequence was obtained by two rounds of PCR, and vector homology arms were added to both ends of the sequence.

[0269] 2) First PCR Step 1: The reaction system was prepared as shown in Table 3.

[0270] [Table 3] Step 2: PCR conditions are shown in Table 4.

[0271] [Table 4] The PCR product was subjected to gel electrophoresis, and the target 0.7 kb band was excised and recovered.

[0272] 3) Second PCR Step 1: The reaction system was prepared as shown in Table 5.

[0273] [Table 5] Step 2: PCR conditions are shown in Table 6.

[0274] [Table 6] The PCR product was subjected to gel electrophoresis, and the 400 bp target band was excised and recovered. 4) Vector PCR

[0275] The vector region of the phagemid was obtained by PCR and used to express the VHH sequences.

[0276] Step 1: The reaction system was prepared as shown in Table 7.

[0277] [Table 7] Step 2: PCR conditions are shown in Table 8.

[0278] [Table 8] The PCR product was subjected to gel electrophoresis, and the target 4000 bp band was excised and recovered.

[0279] 5) Ligation, purification and concentration of ligation products

[0280] The VHH fragments were ligated into a phagemid vector and the ligation products were then concentrated.

[0281] Step 1: The reaction system was prepared as shown in Table 9.

[0282] [Table 9] Step 2: The above mixture was incubated at 50°C for 2 hours and cooled on ice.

[0283] Step 3: The ligated product was purified to remove components such as salt ions and proteins in the ligated system, and concentrated to 1 / 10 of the original volume. 5. Electroporation and Library Construction

[0284] 1) Thaw a tube of competent E. coli on ice.

[0285] 2) 2 μL of the ligation product or positive control was added to the competent E. coli and gently pipetted to homogenize. The mixture was then left on ice for 1-2 minutes, transferred to a pre-chilled electroporation cup, and electroporation was performed.

[0286] 3) Immediately after electroporation, 1 mL of 37°C 2YT-G was added, the electroporation cup was rinsed with a pipette tip, and the electroporated bacterial solution was transferred to a 15 mL centrifuge tube or a 2 mL EP tube and incubated in a 37°C water bath until electroporation of all samples was complete. The 2YT-G (2xYT medium containing 2% glucose) was then transferred to a 37°C shaker and incubated at 220 rpm for 1 hour.

[0287] 4) Aspirate 5 μL of the above bacterial solution and 2 ~10 5 The mixture was diluted 2-fold, plated on 2YT-A plates (2YT plates containing 100 μg / mL ampicillin), and cultured overnight in a 37° C. incubator, and the number of colonies was counted.

[0288] 5) The remaining bacterial solution was inoculated into 2YT-AG medium and shaken until the culture reached the logarithmic growth phase. Helper phage for infection was added, and the culture was shaken at 30°C and 220 rpm for 12 to 16 hours. (2YT-AG: 2xYT medium containing 2% glucose and 100 μg / mL ampicillin.) 6) The phages were collected, concentrated, and titered. 6. Phage Library Antibody Screening

[0289] The phage library obtained in step 7 above was subjected to three rounds of antibody screening, including positive and negative screening. First, the phages were incubated with the antigen peptide, and those that could not bind to the antigen peptide were discarded, leaving only those that bound to the antigen peptide. Next, for negative screening, the phages were incubated with BSA, and only those that could not bind to BSA were retained.

[0290] 1) Coat board The antigen was diluted with PBS to a concentration of 2 ng / μL and added to a 96-well plate at 100 μL / well. 2% BSA was prepared in PBS and added to the corresponding negative screening wells at 100 μL / well. The plate was sealed with plastic wrap and incubated overnight at 4°C.

[0291] 2) The coating solution was discarded, and 200 μL of washing solution (washing solution: 1% Tween 20 / PBS, pH 7.4) was added, followed by washing three times.

[0292] 3) Block 100 μL / well of 2% BSA blocking solution was added to all wells, which were then sealed with plastic wrap and incubated at 37° C. for 1 hour.

[0293] 4) The supernatant was discarded, and 200 μL of washing solution was added and washed three times.

[0294] 5) Add phage (1 x 101) to the positive screening wells. 2 The wells were diluted to 100 μL, sealed with plastic wrap, and incubated at 37° C. for 1 hour.

[0295] 6) The supernatant was discarded, and 200 μL of washing solution was added and washed 10 times. 7) Elution-Neutralization

[0296] 200 μL of eluate was added to the positive screening wells and neutralized to pH 7 to 7.4. 8) Negative screening

[0297] The above eluate was added to the negative screening wells, sealed with plastic wrap, and incubated at 37°C for 1 hour, and the supernatant was aspirated and retained to detect the titer.

[0298] 9) After the first round of panning, a small amount of phage was collected, diluted, and plated onto 2xYT-A plates and incubated overnight at 37°C. The next day, colonies were counted and titers were calculated. Single clones were selected for sequencing to analyze sequence diversity and enrichment.

[0299] 10) All remaining phages were used for TG1 infection. 11) M13KO7 infection

[0300] The phage was diluted, and M13KO7 was added to the bacterial solution, followed by incubation in a water bath at 37°C for 30 minutes. The medium was replaced with 2xYT-AK medium, and the mixture was cultured at 30°C with shaking at 220 rpm for 14 to 16 hours.

[0301] 12) The phages were concentrated and the phage titer was detected, and then the next round of screening was carried out.

[0302] In the second and third rounds of screening, the amount of coated antigen was reduced and the number of washes after incubation of the positive wells with phages was increased, while the other steps were the same as those above. 7. Binding Detection and Sequence Acquisition

[0303] The phages obtained from three rounds of screening were used to infect TG1 with M13KO7 helper phage, and plated onto 2YT-AK plates. Single clones were selected and expanded, and the phages were collected and subjected to binding detection to determine usable phages / antibodies.

[0304] 1) Coat board The antigen was diluted to 1 ng / μL with the coating solution, and 100 μL was added to each well. 2% BSA was added to the negative control wells, which were then sealed with plastic wrap and left at 4°C overnight.

[0305] 2) The coating solution in the plate was discarded, and 200 to 250 μL of washing solution was added to wash the plate three times.

[0306] 3) 200 μL of 2% BSA was added to all wells and blocked for 1 hour at room temperature.

[0307] 4) The blocking solution in the plate was discarded, and 200 μL of washing solution was added to wash the plate once.

[0308] 5) 100 μL of phage was added to each of the positive and negative wells, and the mixture was incubated at 37° C. for 1 hour.

[0309] 6) The phages in the plate were discarded, and the plate was washed three times with 200 μL of washing solution.

[0310] 7) Anti-M13-HRP antibody was diluted to 50 ng / well and incubated at room temperature for 1 hour.

[0311] 8) The antibody in the plate was discarded, and 200 μL of washing solution was added, followed by washing five times.

[0312] 9) 100 μL of TMB coloring solution was added to each well and allowed to react at room temperature until the OD value reached 2-3.

[0313] 10) 50 μL of stopping solution was added to each well of the color development system.

[0314] 11) The absorbance at 450 nm was measured using a spectrophotometer. The monoclonal bacterial solutions corresponding to the positive wells were subjected to sequencing to determine the VHH sequences.

[0315] 12) The obtained antibody was named NLB1-NLB23. 8. Functional screening at the cellular level

[0316] 1) A STAR construct targeting LILRB4 was constructed according to the structure shown in Figure 2. The positive antibody heavy chain (VHH) sequences obtained by the above screening were assembled with the constant region of a STAR molecule and inserted into a lentiviral vector using homologous recombination to construct a complete STAR plasmid. 2) Packaging virus

[0317] 5 × 10 Lentix-293T cells were cultured in a 10 cm culture dish. 5 The cells were inoculated at 1000 cells / mL and cultured in a 37°C, 5% CO2 incubator. Transfection was performed when the cell density reached approximately 80% (as observed under a microscope). The four plasmids were homogenously mixed in 500 μL of serum-free DMEM at a ratio of PMD2.G:PRSV-Rev:PMDlg:transfer plasmid = 1:1:2:4. 54 μL of PEI-max was homogenously mixed with 500 μL of serum-free DMEM and left at room temperature for 5 minutes (a volume-to-mass ratio of PEI-max to plasmid was 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently pipetted, mixed homogenously, and left at room temperature for 15 minutes. The final mixture was slowly added to the medium, mixed homogenously, and then returned to the incubator for an additional 12–16 hours of culture. The medium was then replaced with 6% FBSDMEM medium and further cultured. Virus solutions were collected after 48 and 72 hours. 3) Measurement of virus titer

[0318] TCR knockout Jurkat-C4 cells were plated in a flat-bottom 96-well plate at 1.5 × 10 5 Cells were seeded at 100 μL / mL, and 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000x polybrene was added to each well. The virus was serially diluted 10-fold in 1640 complete medium. 100 μL of the diluted cells were added to the virus wells at a rate of 100 μL / well, mixed, centrifuged at 32°C, 1500 rpm for 90 minutes, and then cultured in a 37°C, 5% CO2 incubator. After 72 hours, the infection efficiency was measured using a flow cytometer. Wells with an infection rate between 2 and 30% were selected to calculate the titer. The formula for titer (TU / mL) was 1.5 x 10. 4 × Positive rate ÷ Virus volume (μL) × 1000. T cells were infected with the above viruses and STAR was expressed. 4) Isolation, activation, and infection of human primary T cells

[0319] Primary T cells were obtained using the Ficoil separation method and cultured at an initial density of 1 x 10 in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2. 6 The cells were cultured at 1000 / mL and activated by adding them to a well plate pre-coated with CD3, CD28, and fibronectin. After 24 hours of activation, the virus solution was added, the cells were centrifuged at 1500 rpm for 90 minutes, and the cells were cultured in a CO2 incubator. 24 hours after infection, X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 was added, and the cells were transferred to another well. Thereafter, the cells were subcultured every 1-2 days. 5) Measurement of killing efficiency by in vitro co-culture of T cells and target cells

[0320] Target THP1 cells or 293T cells overexpressing LILRB4 were seeded in 24-well plates at a density of 1E5 / well and cultured overnight. The corresponding number of STAR-T cells was added to target cells at a STAR-T cell to target cell ratio of 0.5:1. The killing effect of STAR-T cells on target cells was detected after 24 or 48 hours of culture.

[0321] As shown in Figure 3, NLB3, 4, 5, 6, 7, 8, 9, 10, 11, 14, and PC specifically recognized LILRB4 target cells. NLB1, 2, 12, 13, 21, 22, and 23 did not recognize LILRB4. NLB15, 16, 17, 18, 19, and 20 had a nonspecific recognition and killing effect on LILRB4-negative cells. Example 3 Measurement of antibody affinity (SPR)

[0322] Antibody affinity was detected using surface plasmon resonance (SPR) technology. First, a mouse anti-human IgG (Fc) antibody was immobilized on the surface of a CM5 chip by amino coupling. Next, a VHH antibody fused to human IgG1 (Fc) was injected into the capture experimental channel (Fc2). The reference channel (Fc1) did not capture any ligand. LILRB4 protein was serially diluted 2-fold, with a concentration gradient set to 62.5 nM, 31.25 nM, 15.625 nM, 7.813 nM, 3.906 nM, 1.953 nM, and 0.977 nM. The diluted protein was injected sequentially into the experimental and reference channels, allowing binding and dissociation times to correspond. The KD values ​​of the samples were calculated using Biacore 8K analysis software, and the reference channel (Fc1) was used for background subtraction. The results are shown in Table 1 and Figure 4, and the affinities of NLB4 and NLB14 for the LILRB4 protein were 1.32 nM and 0.493 nM, respectively.

[0323] Table 1. Measurement data of affinity between NLB4 and NLB14 [Table 10] Example 4 Nanobody competitive binding measurement (SPR)

[0324] Competitive antibody binding was detected by surface plasmon resonance (SPR) technology. The stationary phase was LILRB4 protein, and the mobile phase was NLB(G4S)IGG1FC. The results are shown in Figures 5A and 5B, and show that NLB4 and NLB14 did not compete with LILRB4 for binding. Example 5: Recognition epitopes of NLB4 / NLB14 nanoantibodies on LILRB4

[0325] The structure of LILRB4 is shown in Figure 6. The epitopes recognized by the NLB4 / NLB14 nanobody on LILRB4 were detected by flow cytometry. The results are shown in Figure 7. NLB4 or NLB14 could recognize the full-length extracellular region of LILRB4, but could not bind to individual Ig domains or the stock region. Example 6 Measurement of antibody binding specificity (SPR)

[0326] In this experiment, to determine whether the antibodies bind nonspecifically to other LILRB4 family proteins (LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, and LILRB5), we first constructed 293T cell lines expressing the same family proteins by lentiviral infection and co-expressed RFP with these proteins. We then determined the infection efficiency by detecting RFP positivity using flow cytometry, and then stained the LILRB4 antibody to detect nonspecific binding to homologous family proteins. The results are shown in Figure 8, demonstrating that two LILRB4 nanoantibodies, NLB4 and NLB14, significantly bound to LILRB4 but not to other proteins in the same family. Example 7 Cell line binding measurements for LILRB4 safety assessment

[0327] In this experiment, we constructed a safety evaluation cell library consisting of nine cell lines derived from different human tissues (lung, muscle, blood vessels, prostate, liver, kidney, brain, intestine, and skin) to assess the accuracy of target recognition by the two antibodies, NLB4 and NLB14, and to detect nonspecific binding to other components of human tissues. We then performed flow cytometry analysis to preliminary evaluate the safety of the antibodies. The NLB4 and NLB14 antibodies fused with the FC (human IGG1FC) tag were incubated with the safety evaluation cell lines to allow binding, and then labeled with an anti-FC fluorescent secondary antibody and analyzed by flow cytometry. The results are shown in Figures 9A and 9B. NLB4 and NLB14 did not exhibit nonspecific binding to any of the nine tissue cell lines. Example 8 Evaluation of species specificity of NLB4 and NLB14 nanobodies - mice

[0328] In this example, we established 293T-human LILRB4 and 293T-mouse LILRB4 antibodies and used flow cytometry to detect the species specificity of the two antibodies, NLB4 and NLB14. The results are shown in Figure 10. The NLB4 and NLB14 antibodies recognized only human LILRB4, but neither recognized mouse LILRB4. Example 9 Structure and membrane expression of single-epitope and dual-epitope LILRB4 STAR

[0329] To detect the membrane expression, in vitro killing effect, and in vivo killing effect of single-epitope and dual-epitope LILRB4 STAR, STAR structures targeting LILRB4 were constructed as shown in Figure 11, where the NLB4 STAR amino acid sequence is shown in SEQ ID NO:39, the NLB14 STAR amino acid sequence is shown in SEQ ID NO:40, the NLB4 / NLB14 STAR amino acid sequence is shown in SEQ ID NO:41, and the NLB4 / (myc)NLB14 STAR amino acid sequence is shown in SEQ ID NO:42.

[0330] For STAR-T cells, primary T cells were infected with lentivirus and membrane expression was detected using flow cytometry. RFP fluorescent protein was co-expressed at the end of STAR via an IRES, and the membrane status of the dual-epitope STAR was determined using an anti-mouse TCR β chain antibody and RFP expression efficiency. The experimental results are shown in Figure 12. STAR showed very high infection efficiency and was double-positive for mouse TCR β chain and RFP, indicating that STAR can be expressed on the membrane. After confirming membrane expression, the IRES-RFP sequence in the vector was deleted. Example 10 Killing activity and killing specificity of LILRB4 STAR-T cells against the target cell line THP1

[0331] To verify the specific killing of LILRB4 target cells by STAR-T, we stably overexpressed firefly luciferase in the AML cell line THP1. Based on this, we knocked out the LILRB4 gene in THP1 cells using CRISPR (Figure 13A). We screened for nanoantibodies NLB4 and NLB14 that recognize LILRB4 using alpaca mice. NLB4, NLB14, and NLB4 / NLB14 were simultaneously constructed into STAR vectors to obtain LILRB4-1 STAR, LILRB4-2 STAR, and dual-epitope STAR (biparatopic STAR). These STARs were then introduced into T cells via the lentiviral vector to obtain LILRB4-1 STAR-T, LILRB4-2 STAR-T, and LILRB4 dual-epitope STAR-T. To evaluate the function of these STAR-Ts, we co-cultured STAR-T and target cells for 24 hours, followed by the addition of luciferase substrate, and the killing effect of STAR-T on target cells was detected by chemiluminescence. The experimental results are shown in Figure 13. LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual epitope STAR-T had potent killing effects only on THP1 cells, but not on THP1-LILRB4KO cells. This indicates that LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual epitope STAR-T can specifically recognize and kill LILRB4 target cells. Example 11 Killing Effect of LILRB4 STAR-T Cells on Various AML Cell Lines

[0332] Two simple epitope STAR-T and dual epitope STAR-T were used to detect the killing of four target cells. As shown in Figure 14A, LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual epitope STAR-T cells all exhibited potent killing activity against the high-antigen-density AML target cell lines THP1, MV-11, and OCI-AML3 cells after 24 hours of coculture. However, LILRB4 dual epitope STAR-T showed no clear advantage over single epitope STAR-T. As shown in Figure 14B, after 6, 15, and 24 hours of coculture, LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual epitope STAR-T cells also exhibited potent killing activity against KASUMI-1, a cell line with low LILRB4 expression. Furthermore, the killing efficiency of LILRB4 dual-epitope STAR-T against target cells with low antigen density was significantly higher than that of single-epitope STAR-T, suggesting that dual-epitope STAR-T is more effective at eliminating target cells with low LILRB4 expression.The results of in vitro killing experiments showed that dual-epitope STAR-T can efficiently and specifically kill LILRB4 target cells at the in vitro cellular level. Example 12 Cytokine secretion levels of LILRB4 STAR-T cells

[0333] After co-culture of LILRB4 STAR-T cells with MV4-11 target cells for 24 hours, culture supernatants were collected and assayed for the cytokines IL-2, IFN-γ, and TNF-α. The results are shown in Figure 15. After co-culture with MV4-11 target cells, both single-epitope and dual-epitope STAR-T significantly increased IL-2 and IFN-γ cytokine levels, but TNF-α levels were relatively low. This suggests that dual-epitope STAR-T may be safer for clinical applications because TNF-α can directly stimulate macrophages, neutrophils, and other cells to secrete IL-6, and IL-6 is the primary cytokine responsible for cytokine storms in cell therapy. Furthermore, dual-epitope STAR-T secreted significantly more IL-2 and IFN-γ than LILRB4-1 STAR-T and LILRB4-2 STAR-T, suggesting that dual-epitope STAR-T may have a more efficient tumor-killing effect. Example 13 Evaluation of the killing effect of LILRB4 STAR-T on a mouse tumor model

[0334] To verify the in vivo killing activity and potential safety issues of STAR-T cells, we constructed an intravital imaging model of MV4-11 NCG mouse tumors by intravenously injecting luciferase-expressing MV4-11 cells into NCG immunodeficient mice (tail vein injection, 1E6 / mouse). Furthermore, to verify the tumor-killing effect of dual-epitope STAR-T, we selected an antibody scFv published by Texas A&M and constructed STAR-T as a positive control (PCSTAR-T). Seven days after MV4-11 tumor cell injection, LILRB4-1 STAR-T cells, LILRB4-2 STAR-T cells, dual-epitope STAR-T cells, control T cells (PCSTAR-T), and mock-T cells (T cells not infected with STAR) were injected into the tail vein at 4E6 cells per mouse. Intravital imaging was performed to detect tumor growth from day 7 onward, twice weekly in the early stage and once weekly in the late stage. The results are shown in Figures 16A and 16B. Dual-epitope STAR-T exhibited significant tumor growth inhibitory activity in mouse tumor models, and was significantly superior to the PC positive control group, LILRB4-1 STAR-T, and LILRB4-2 STAR-T groups. Example 14 Experimental data on the safety of LILRB4 STAR-T cells

[0335] Regarding animal safety, we monitored mouse behavior (e.g., appearance, behavior, response to stimuli, feces, etc.), mortality, and weight changes before and after cell injection. In the STAR-T cell group, there was no change in mouse appearance (e.g., fur or color) before and after STAR-T cell injection. Mouse behavior was normal, there was no sudden increase in sensitivity to stimuli, and there was no significant change in feces (observation, not quantification). As shown in Figure 16A, mortality data indicated that animals in the Mock-T group died on day 28 due to a sustained increase in tumor burden, while animals in the STAR-T group did not die until day 34. Furthermore, as shown in Figure 16C, reinjection of STAR-T did not significantly affect mouse weight, indicating a high level of safety. Example 15 In vivo pharmacokinetics study of LILRB4 STAR-T cells

[0336] To examine the in vivo metabolism of STAR-T cells, we first collected blood from the orbits of mice and examined their in vivo proliferation. The results are shown in Figure 16D. After injection, dual-epitope STAR-T cells showed rapid proliferation, with the proliferation rate significantly higher than that of the PC control group. However, once tumor cells died, the STAR-T cells subsequently showed a downward trend and remained at a low level. Furthermore, the number of dual-epitope STAR-T cells in the peripheral blood of mice was significantly higher than that of the other groups, especially on days 10 and 14 after T cell reinfusion, indicating that STAR-T exerted a superior proliferation effect in mice. Example 16 In vivo systemic toxicity experiment of LILRB4 STAR-T cells

[0337] To further evaluate the safety of LILRB4 STAR-T in mice, STAR-T and Mock-T cells were injected into the tail vein at an effective cell dose of 4E6 per mouse 7 days after the reinfusion of luciferase-expressing MV4-11 cells. On days 14 and 28 after the reinfusion of T cells, the mouse heart, liver, spleen, lungs, kidneys, small intestine, pancreas, and brain were harvested and fixed, and HE staining was performed to examine the pathological changes in the mouse tissues. Results showed that no obvious lesions were observed in the mouse tissues 14 and 28 days after the reinfusion of STAR-T (Figure 17). Example 17 Detection of the in vitro tumorigenicity of LILRB4 STAR-T cells

[0338] To verify the tumorigenic potential of LILRB4 STAR-T cells, soft agar clonal growth assays were performed on LILRB4 STAR-T cells. 0.5 × 10 LILRB4 STAR-T cells were cultured in a 200-well plate. 3 , 1.0X10 3 , 2.0X10 3 The AML tumor cell line OCI-AML3 was administered at 1.0 × 10 3 As a positive control, a dose of 2.0 × 10 was used. As a negative control, Mock-T (T cells not infected with STAR, dose 2.0 × 10) 3) were used. The above groups of cells were subjected to clonal growth experiments on 0.6% bottom and 0.35% top soft agar, respectively. Observation times were 8, 15, and 21 days after cell seeding. The observation results are shown in Figure 18. OCI-AML3 tumor cells (positive control group) formed small clones on day 8 after seeding and large clones on day 21. However, the three doses of LILRB4 STAR-T cells and Mock-T cells did not form clones until day 21. The above results indicate that LILRB4 STAR-T cells were unable to form clones in the soft agar cloning experiment, suggesting that they do not have tumorigenic potential in vitro. Example 18 Relationship between NLB4 / NLB14 and LILRB4 ligand ApoE

[0339] LILRB4 binds to ApoE and supports tumor cell invasion into tissues and inhibits T cell activity in acute myeloid leukemia (AML) cells via the ApoE-LILRB4-SHP-2-uPAR-Arginase-1 signaling pathway. Competitive binding of NLB4 / NLB14 to ApoE was detected using LILRB4 protein as the stationary phase, ApoE as mobile phase 1, and NLB(G4S)IGG1FC as mobile phase 2. The results are shown in Figure 19A. NLB4 / NLB14 and ApoE did not compete for binding to LILRB4, and ApoE did not affect the binding of NLB4 / NLB14 to LILRB4. Flow cytometric analysis of the target cell line, KASUMI-1, is shown in Figure 19B. Among AML cell lines, the myeloid line, KASUMI-1, did not express ApoE. The ApoE concentration in normal peripheral blood was 29–70 ng / μL. Thus, we detected the killing effects of single-epitope STAR-T and dual-epitope STAR-T on target cells at ApoE concentrations of 0, 40 ng / μL, and 80 ng / μL. The results are shown in Figure 19C, and ApoE did not inhibit target cell killing by LILRB4 STAR-T.

[0340] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the technical concept of the present invention, and all of these simple modifications also fall within the protection scope of the present invention.

[0341] Furthermore, the various specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, the present invention will not further describe the various possible combinations.

[0342] Sequences according to the present application: SEQ ID NO:1 Amino acid sequence of wild-type human TCR alpha constant region DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* SEQ ID NO:2 Amino acid sequence of wild-type human TCR beta constant region DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF* SEQ ID NO:3 Amino acid sequence of wild-type murine TCR alpha constant region DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO:4 Amino acid sequence of wild-type murine TCR beta constant region DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS SEQ ID NO:5 Mouse T cell receptor alpha chain constant region containing a cysteine ​​substitution (MouseTCRaC-Cys) DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO:6 Mouse T cell receptor beta chain constant region containing a cysteine ​​substitution (MouseTCRβC-Cys) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS SEQ ID NO:7 Mouse T cell receptor alpha chain constant region containing hydrophobic amino acid substitutions (mouseTCRaC-TM9) DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO:8 Mouse T cell receptor alpha chain constant region containing a lysine substitution in the transmembrane region (mouseTCRaC-Argmut) DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLRVAGFNLLMTLRLWSS SEQ ID NO:9 Constant region of mouse T cell receptor beta chain containing lysine substitutions in the transmembrane and intracellular regions (mouseTCRβC-Argmut); DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGRATLYAVLVSTLVVMAMVRRRNS SEQ ID NO:10 CD40 intracellular domain amino acid sequence KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISV SEQ ID NO:11 OX40 intracellular domain amino acid sequence RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO:12 ICOS intracellular domain amino acid sequence KKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID NO:13 CD28 intracellular domain amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:14 4-1BB intracellular domain amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO:15 CD27 intracellular domain amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO:16 Mouse T cell receptor alpha chain constant region deleted with cysteine ​​substitutions and altered hydrophobic regions (mouseTCRαC-delmut) DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLW SEQ ID NO:17 Mouse T cell receptor beta chain constant region with deleted intracellular domain and containing cysteine ​​substitutions (mouseTCRβC-delmut) DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAM SEQ ID NO:18 Human CD3γ amino acid sequence MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN SEQ ID NO:19 Human CD3δ amino acid sequence MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK SEQ ID NO:20 Human CD3ε amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS KPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI SEQ ID NO:21 Human CD3 zeta amino acid sequence MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:22 Human IL-2β receptor intracellular terminal amino acid sequence NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO:23 Human IL-7α receptor intracellular terminal amino acid sequence KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNQ SEQ ID NO:24 Human IL-21 receptor intracellular terminal amino acid sequence SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCE DDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS SEQ ID NO:25 Amino acid sequence of human STAT5 activation module YRHQ SEQ ID NO:26 Human IL-2β receptor intracellular tail and human STAT5 activation module amino acid sequence IL-2RbQ NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVGGGGSYRHQ SEQ ID NO:27 Human IL-7α receptor intracellular tail and human STAT5 activation module amino acid sequence IL-7RbQ KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNQGGGGSYRHQ SEQ ID NO: 28 VHH amino acid sequence of LILRB4 antibody NLB4 DVQLQESGGGLVQSGGSLRLSCLASGTSGNVKAVGWYGWYRQAPGKQREVVATITRGGIPNYADSVQGRFTISRDNAQDTVFLQMNSLKPADTAVYYCYARILTDDWHDLWGQGTQVTVSS SEQ ID NO: 29 VHH amino acid sequence of LILRB4 antibody NLB14 DVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCVSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQYSSTWTIRLTRCHFGSWGQGTQVTVSS SEQ ID NO: 30 mouseTCRaC-Cys-TM9 (hmctSTARTCRaC) amino acid sequence DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO:31 mouseTCRaC-Cys-TM9-N.Rec (NrecSTARTCRaC) amino acid sequence DIQNPDPAVYQLRDDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 32 Mouse T cell receptor beta chain constant region (MouseTCRbC-Cys-N.Rec, NrecSTARTCRbC) amino acid sequence including N-terminal modifications and cysteine ​​substitutions DLKNVFPPEVAVFEPSAEIATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS SEQ ID NO:33 anti-LILRB4NLB4CDR1 amino acid sequence GTSGNVKAVG SEQ ID NO:34 anti-LILRB4NLB4CDR2 amino acid sequence TITRGGIPN SEQ ID NO:35 anti-LILRB4NLB4CDR3 amino acid sequence RILTDDWHDL SEQ ID NO:36 anti-LILRB4NLB14CDR1 amino acid sequence GFTLDYYAIG SEQ ID NO:37 anti-LILRB4NLB14CDR2 amino acid sequence CVSSSDGSTY SEQ ID NO:38 anti-LILRB4NLB14CDR3 amino acid sequence DQYSSTWTIRLTRCHFGS SEQ ID NO:39 NLB4 STAR amino acid sequence * SEQ ID NO:40 NLB14 STAR amino acid sequence MLLLVTSLLLCELPHPAFLLIPDVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCVSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQYSSTWTIRLTRCHFGSWGQGTQVTVSSEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWGGGGSGGGGSGGGGSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI* SEQ ID NO:41 NLB4 / NLB14 STAR MLLLVTSLLLCELPHPAFLLIPDVQLQESGGGLVQSGGSLRLSCLASGTSGNVKAVGWYGWYRQAPGKQREVVATITRGGIPNYADSVQGRFTISRDNAQDTVFLQMNSLKPADTAVYYCYARILTDDWHDLWGQGTQVTVSSEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCVSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQYSSTWTIRLTRCHFGSWGQGTQVTVSSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWGGGGSGGGGSGGGGSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI* SEQ ID NO:42 NLB4 / (myc)NLB14 STAR * SEQ ID NO:43 Constant region of the murine T-cell receptor alpha chain with a deleted intracellular domain and containing N-terminal modifications, cysteine ​​substitutions, and hydrophobic changes in the transmembrane domain DIQNPDPAVYQLRDDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLW SEQ ID NO:44 Mouse T-cell receptor beta chain constant region with deleted intracellular domain and containing N-terminal modifications and cysteine ​​substitutions DLKNVFPPEVAVFEPSAEIATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAM SEQ ID NO:45 Amino acid sequence of the constant region of the wild-type human TCR gamma chain: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVVYFAIITCCLLRRTAFCCNGEKS SEQ ID NO:46 Amino acid sequence of the constant region of the wild-type murine TCR gamma chain: XKRLDADISPKPTIFLPSVAETNLHKTGTYLCLLEKFFPDVIRVYWKEKDGNTILDSQEGDTLKTNDTYMKFSWLTVPERAMGKEHRCIVKHENNKGGADQEIFFPSIKKVAVSTKPTTCWQDKNDVLQLQFTITSAYYTYLLLLLKSVIYLAIISFSLLRRTSVCGNEKKS SEQ ID NO:47 Amino acid sequence of the constant region of the wild-type human TCR δ chain: XSQPHTCPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL* SEQ ID NO:48 Amino acid sequence of the constant region of the wild-type murine TCR delta chain: XSQPPAKPSVFIMKNGTNVACLVKDFYPKEVTISLRSSKIVEFDPAIVISPSGKYSAVKLGQYGDSNSVTCSVQHNSETVHSTDFEPYANSFNNEKLPEPENDTQISEPCYGPRVTVHTEKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF* SEQ ID NO:49 EAAAK linker amino acid sequence EAAAK SEQ ID NO:50 (EAAAK)2 linker amino acid sequence EAAAKEAAAK SEQ ID NO:51 (EAAAK)3 linker amino acid sequence EAAAKEAAAKEAAAK SEQ ID NO:52 EAAK linker amino acid sequence EAAK SEQ ID NO:53 (EAAK)2 linker amino acid sequence EAAKEAAK SEQ ID NO:54 (EAAK)3 linker amino acid sequence EAAAKEAAKEAAK SEQ ID NO:55 A2(EAAAK)2A linker amino acid sequence AAEAAAKEAAAKA SEQ ID NO:56 A3(EAAAK)3A linker amino acid sequence AAAEAAAKEAAAKEAAAKA SEQ ID NO:57 A4(EAAAK)4A linker amino acid sequence AAAAAAAKEAAAKA SEQ ID NO:58 A5(EAAAK)5A linker amino acid sequence AAAAAEAAAKEAAAKA SEQ ID NO:59 A(EAAAK)4ALEA(EAAAK)4A linker amino acid sequence AEAAAKEAAAKAAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA SEQ ID NO:60 G4S2 linker amino acid sequence GGSGGS SEQ ID NO:61 (G4S2)2 linker amino acid sequence GGSGGSGGSGGS SEQ ID NO:62 (G4S2)3 linker amino acid sequence GGSGGSGGSGGSGGSGGS SEQ ID NO:63 (G4S2)4 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:64 (G4S2)5 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:65 (G4S2)6 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:66 (G4S2)7 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:67 (G4S2)8 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:68 (G4S2)9 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:69 (G4S2)10 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:70 S7 linker amino acid sequence SSSSSSS SEQ ID NO:71 G3S linker amino acid sequence GGGS SEQ ID NO:72 (G3S)2 linker amino acid sequence GGGSGGGS SEQ ID NO:73 (G3S)3 linker amino acid sequence GGGSGGGSGGGS SEQ ID NO:74 (G3S)4 linker amino acid sequence GGGSGGGSGGGSGGGS SEQ ID NO:75 (G3S)5 linker amino acid sequence GGGSGGGSGGGSGGGSGGGS SEQ ID NO:76 (G3S)6 linker amino acid sequence GGGSGGGSGGGSGGGSGGGSGGGS SEQ ID NO:77 (G3S)7 linker amino acid sequence GGGSGGGSGGGSGGGSGGGSGGGSGGGS SEQ ID NO:78 (G3S)8 linker amino acid sequence GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS SEQ ID NO:79 (G3S)9 linker amino acid sequence GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS SEQ ID NO:80 (G3S)10 linker amino acid sequence GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS SEQ ID NO:81 G4S linker amino acid sequence GGGGS SEQ ID NO:82 (G4S)2 linker amino acid sequence GGGGSGGGGS SEQ ID NO:83 (G4S)3 linker amino acid sequence GGGGSGGGGSGGGGS SEQ ID NO:84 (G4S)4 linker amino acid sequence GGGGSGGGGSGGGGSGGGGS SEQ ID NO:85 (G4S)5 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:86 (G4S)6 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:87 (G4S)7 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:88 (G4S)8 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:89 (G4S)9 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:90 (G4S)10 linker amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:91 G2S linker amino acid sequence GGS SEQ ID NO:92 (G2S)2 linker amino acid sequence GGSGGS SEQ ID NO:93 (G2S)3 linker amino acid sequence GGSGGSGGS SEQ ID NO:94 (G2S)4 linker amino acid sequence GGSGGSGGSGGS SEQ ID NO:95 (G2S)5 linker amino acid sequence GGSGGSGGSGGSGGS SEQ ID NO:96 (G2S)6 linker amino acid sequence GGSGGSGGSGGSGGSGGS SEQ ID NO:97 (G2S)7 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGS SEQ ID NO:98 (G2S)8 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:99 (G2S)9 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:100 (G2S)10 linker amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO:101 GS linker amino acid sequence GS SEQ ID NO:102 (GS)2 linker amino acid sequence GSGS SEQ ID NO:103 (GS)3 linker amino acid sequence GSGSGS SEQ ID NO:104 (GS)4 linker amino acid sequence GSGSGSGS SEQ ID NO:105 (GS)5 linker amino acid sequence GSGSGSGSGS SEQ ID NO:106 (GS)6 linker amino acid sequence GSGSGSGSGSGS SEQ ID NO:107 (GS)7 linker amino acid sequence GSGSGSGSGSGSGS SEQ ID NO:108 (GS)8 linker amino acid sequence GSGSGSGSGSGSGSGS SEQ ID NO:109 (GS)9 linker amino acid sequence GSGSGSGSGSGSGSGSGS SEQ ID NO:110 (GS)10 linker amino acid sequence GSGSGSGSGSGSGSGSGSGS SEQ ID NO:111 (G)5 linker amino acid sequence GGGGG SEQ ID NO:112 (A)11 linker amino acid sequence AAAAAAAAA SEQ ID NO:113 F2A cleavable linker amino acid sequence VKQTLNFDLLKLAGCVESNPG SEQ ID NO:114 P2A cleavable linker amino acid sequence GSGATNFSLLKQAGDVEENPGP SEQ ID NO:115 T2A cleavable linker amino acid sequence EGRGSLLTCGDVEENPG SEQ ID NO:116 E2A cleavable linker amino acid sequence QCTNYALLKLAGDVESNPG SEQ ID NO:117 Disulfide bond type cleavable linker amino acid sequence LEAGCKNFFPRSFTSCGSLE SEQ ID NO:118 NLB4 VHH nucleotide sequence GATGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTTCAATCTGGAGGCAGCCTGAGGCTGAGCTGTCTGGCCAGCGGAACCTCCGCAACGTGAAGGCCGTGGGCTGGTACGGCTGGTATAGACAGGCCCCTGGCAAGCAGCGGGAAGTGGTGGCCACAATCACCAGAGGCGGCATCCCCA ATTACGCCGATAGCGTGCAAGGCAGATTCACCATCAGCCGGGACAACGCCCAGGACACAGTGTTCCTGCAGATGAACAGCCTGAAACCTGCTGATACCGCCGTGTACTACTGCTACGCCAGAATCCTGACAGACGACTGGCACGACCTGTGGGGCCAGGGCACCCAGGTGACCGTGTCCAGC SEQ ID NO:119 NLB14 VHH nucleotide sequence GACGTCCAGCTGCAGGAGAGCGGCGGAGGACTGGTTCAACCTGGAGGCAGCCTGAGACTGAGCTGTGCTGCTTCTGGCTTTACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCTGGCAAGGAACGGGAAGGCGTGTCCTGCGTGTCCAGCAGTGATGGCTCTACCTACTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAATACCGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACCGCCGTGTACTACTGCGCCGCCGACCAGTACAGCTCTACATGGACCATCCGGCTGACAAGATGCCACTTCGGCAGCTGGGGCCAGGGCACACAGGTGACCGTGTCTAGC SEQ ID NO:120 Nucleotide sequence of NLB4 STAR SEQ ID NO:121 NLB14 STAR nucleotide sequence SEQ ID NO:122 NLB4 / NLB14 STAR nucleotide sequence SEQ ID NO:123 NLB4 / (myc)NLB14 STAR nucleotide sequence

Claims

1. A synthetic T cell receptor antigen receptor, the synthetic T cell receptor antigen receptor comprises an α chain and a β chain, the α chain comprising a first target binding region and a first constant region, and the β chain comprising a second target binding region and a second constant region, or the α chain comprising a first target binding region and the β chain comprising a second target binding region and a second constant region, or ii) the synthetic T cell receptor antigen receptor comprises a gamma chain and a delta chain, wherein the gamma chain comprises a first target binding region and a first constant region and the delta chain comprises a second target binding region and a second constant region, or wherein the gamma chain comprises a first target binding region and a first constant region and the delta chain comprises a second target binding region and a second constant region; the first target binding region and / or the second target binding region comprises one or more antigen binding regions, the antigen binding regions being the same or different, and the antigen binding regions being directly linked or linked via a linker; A synthetic T cell receptor antigen receptor, characterized in that the antigen-binding region in the first target binding region comprises an antibody or antibody fragment that specifically binds LILRB4, and the antigen-binding region in the second target binding region comprises an antibody or antibody fragment that specifically binds LILRB4.

2. the antigen-binding region of the first target binding region comprises a single chain antibody or a single domain antibody that specifically binds LILRB4, and / or the antigen-binding region of the second target binding region comprises a single chain antibody or a single domain antibody that specifically binds LILRB4; Preferably, the single chain antibody comprises a heavy chain variable region and a light chain variable region that are linked directly or via a linker; 2. The synthetic T cell receptor antigen receptor of claim 1, wherein preferably, the multiple antigen-binding regions in the first target-binding region and / or the second target-binding region bind different regions of LILRB4, e.g., different epitopes.

3. the antigen-binding region of the first target binding region comprises one or more single domain antibodies, and / or the antigen-binding region of the second target binding region comprises one or more single domain antibodies; Preferably, the single domain antibodies comprised in the antigen-binding region of said first target-binding region are identical or different; Preferably, the single domain antibodies comprised in the antigen-binding region of said second target-binding region are identical or different; More preferably, the multiple single domain antibodies are linked directly or via a linker.

4. the single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising CDR1-3; i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

5. 4. The synthetic T cell receptor antigen receptor of claim 3, wherein the single domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

6. i) the α chain and / or the β chain have at least one functional domain linked to their C-terminus, said at least one functional domain being linked to the C-terminus of the α chain and / or the β chain directly or via a linker; or, ii) the gamma and / or delta chains have at least one functional domain linked to their C-terminus, and said at least one functional domain is linked to the C-terminus of the gamma and / or delta chain directly or via a linker.

7. i) the intracellular regions of the α and / or β chains of said synthetic T cell receptor antigen receptor are deleted; or, ii) The synthetic T cell receptor antigen receptor of claim 1, wherein the intracellular regions of the gamma and / or delta chains of the synthetic T cell receptor antigen receptor are deleted.

8. i) the α chain and / or the β chain have at least one functional domain linked to their C-terminus, said at least one functional domain being linked to the C-terminus of the α chain and / or the β chain directly or via a linker; or, ii) the gamma and / or delta chains have at least one functional domain linked to their C-terminus, said at least one functional domain being linked to the C-terminus of the gamma and / or delta chain either directly or via a linker.

9. i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the alpha chain of the synthetic T cell receptor antigen receptor, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the beta chain of the synthetic T cell receptor antigen receptor; or, ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the gamma chain of the synthetic T cell receptor antigen receptor, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are linked to the C-terminus of the delta chain of the synthetic T cell receptor antigen receptor; A synthetic T cell receptor antigen receptor according to any one of claims 6 to 8, characterized in that the functional domains are the same or different.

10. the functional domain comprises a costimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof; Preferably, the costimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB, or CD27; Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, LAG3; Preferably, the cytokine receptor is selected from an interleukin receptor, an interferon receptor, a tumor necrosis factor superfamily receptor, a colony-stimulating factor receptor, a chemokine receptor, a growth factor receptor, or other membrane protein; A synthetic T cell receptor antigen receptor according to any one of claims 6 to 8, characterized in that the intracellular protein is preferably a T cell regulatory factor, such as a NIK domain.

11. the linker is selected from a rigid linker, a flexible linker, a cleavable linker or a nonsense amino acid; Preferably, the amino acid sequence of the rigid linker is one or more selected from SEQ ID NO: 49 to 59; Preferably, said flexible linker is selected from glycine and / or serine rich peptide fragments, preferably said flexible linker is one or more selected from SEQ ID NO: 60-112; 11. The synthetic T cell receptor antigen receptor of any one of claims 1 to 10, wherein the cleavable linker is preferably one or more selected from SEQ ID NOs: 113 to 117.

12. the first constant region is a TCR alpha chain constant region or a TCR gamma chain constant region, preferably a modified TCR alpha chain constant region or a modified TCR gamma chain constant region; Preferably, the constant region of the TCR alpha chain is selected from the constant region of a human TCR alpha chain or the constant region of a murine (preferably mouse) TCR alpha chain; 12. A synthetic T cell receptor antigen receptor according to any one of claims 1 to 11, wherein preferably the constant region of the TCRγ chain is selected from the constant region of the human TCRγ chain or the constant region of the murine (preferably mouse) TCRγ chain.

13. the modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region and comprises one or more modifications at positions 6, 13, 15-18, 48, 112, 114, 115 relative to the wild-type murine (preferably mouse) TCR alpha chain constant region, wherein the modifications are mutations or deletions; or 13. The synthetic T cell receptor antigen receptor of claim 12, wherein the modified TCR alpha chain constant region is derived from the murine (preferably mouse) TCR alpha chain constant region and comprises one or more modifications at positions 13, 36, 47, 53, 58, 78, 98, 122 relative to the wild-type murine (preferably mouse) TCR alpha chain constant region, wherein the modifications are mutations or deletions.

14. the modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably mouse) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., a threonine T to a cysteine ​​C; the modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably mouse) TCR alpha chain constant region, a change at amino acid position 112, e.g., serine S, to leucine L; a change at amino acid position 114, e.g., methionine M, to isoleucine I; and / or a change at amino acid position 115, e.g., glycine G, to valine V; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of amino acid at position 6, e.g., E with D, a substitution of K with R at position 13, and a deletion of amino acids at positions 15-18; the modified TCR alpha chain constant region is derived from the murine (preferably mouse) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably mouse) TCR alpha chain constant region, a substitution of amino acid K at position 122 by R; the modified TCR alpha chain constant region is derived from a murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., threonine T, to cysteine ​​C; a change at amino acid position 112, e.g., serine S, to leucine L; a change at amino acid position 114, e.g., methionine M, to isoleucine I; and a change at amino acid position 115, e.g., glycine G, to valine V; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., threonine T to cysteine ​​C, and a substitution of amino acid K at position 122 by R; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, an amino acid at position 6, e.g., a substitution of E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15-18, and a mutation of amino acid at position 48, e.g., threonine T to cysteine ​​C; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a mutation at amino acid position 48, e.g., threonine T, to cysteine ​​C; a change at amino acid position 112, e.g., serine S, to leucine L; a change at amino acid position 114, e.g., methionine M, to isoleucine I; a change at amino acid position 115, e.g., glycine G, to valine V; and a substitution of amino acid K at position 122 with R; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of the amino acid at position 6, e.g., E with D, a substitution of K at position 13 with R, a deletion of amino acids at positions 15-18, a mutation of the amino acid at position 48, e.g., threonine T, to cysteine ​​C, a change of the amino acid at position 112, e.g., serine S, to leucine L, a change of the amino acid at position 114, e.g., methionine M, to isoleucine I, a change of the amino acid at position 115, e.g., glycine G, to valine V, and a substitution of the amino acid at position 122, e.g., K, with R; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of amino acid at position 6, e.g., E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15-18, a mutation of amino acid at position 48, e.g., threonine T to cysteine ​​C, a change of amino acid at position 112, e.g., serine S to leucine L, a change of amino acid at position 114, e.g., methionine M to isoleucine I, and a change of amino acid at position 115, e.g., glycine G to valine V; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of the amino acid at position 6, e.g., E with D, a substitution of K at position 13 with R, a deletion of amino acids at positions 15-18, a mutation of the amino acid at position 48, e.g., threonine T to cysteine ​​C, and a substitution of amino acid K at position 122 with R; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, an amino acid at position 6, e.g., a substitution of E with D, a substitution of K with R at position 13, a deletion of amino acids at positions 15-18, a change of an amino acid at position 112, e.g., serine S, to leucine L, a change of an amino acid at position 114, e.g., methionine M, to isoleucine I, and a change of an amino acid at position 115, e.g., glycine G, to valine V; the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of amino acid at position 6, e.g., E by D, a substitution of K by R at position 13, a deletion of amino acids at positions 15-18, and a substitution of amino acid K by R at position 122; or 13. The synthetic T cell receptor antigen receptor of claim 12, wherein the modified TCR alpha chain constant region is derived from the murine (preferably murine) TCR alpha chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR alpha chain constant region, a substitution of the amino acid at position 6, e.g., E with D, a substitution of K at position 13 with R, a deletion of amino acids at positions 15-18, a change of the amino acid at position 112, e.g., serine S, to leucine L, a change of the amino acid at position 114, e.g., methionine M, to isoleucine I, a change of the amino acid at position 115, e.g., glycine G, to valine V, and a substitution of the amino acid at position 122 with R.

15. The synthetic T cell receptor antigen receptor of claim 12, wherein the first constant region comprises an amino acid sequence set forth in one of SEQ ID NOs: 1, 3, 5, 7, 8, 16, 30, 31, or 43.

16. the second constant region is a TCR β chain constant region or a TCR δ chain constant region, preferably a modified TCR β chain constant region or a modified TCR δ chain constant region; Preferably, the constant region of the TCR β chain is selected from the constant region of a human TCR β chain or the constant region of a murine (preferably mouse) TCR β chain; 16. A synthetic T cell receptor antigen receptor according to any one of claims 1 to 15, wherein preferably the constant region of the TCRδ chain is selected from the constant region of the human TCRδ chain or the constant region of the murine (preferably mouse) TCRδ chain.

17. the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region and comprises one or more modifications at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170 relative to the wild-type murine (preferably mouse) TCR β chain constant region, wherein the modifications are mutations or deletions; or 17. The synthetic T cell receptor antigen receptor of claim 16, wherein the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region and comprises one or more modifications at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, 170 relative to the wild-type murine (preferably mouse) TCR β chain constant region, wherein the modifications are mutations or deletions.

18. the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region and comprises a mutation at amino acid position 56, e.g., serine S to cysteine ​​C, relative to the wild-type murine (preferably mouse) TCR β chain constant region; the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region and comprises a substitution of lysine at position 150, 168, or 170 with arginine; the modified TCR β chain constant region is derived from the murine (preferably murine) TCR β chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR β chain constant region, a substitution of an amino acid at position 3, e.g., R with K, a substitution of an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, and deletions of amino acids at positions 17, 21-25; the modified TCR β chain constant region is derived from a murine (preferably murine) TCR β chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR β chain constant region, a mutation at amino acid position 56, e.g., serine S to cysteine ​​C, and a substitution of lysine at position 150, 168, or 170 with arginine; the modified TCR β chain constant region is derived from the murine (preferably murine) TCR β chain constant region and comprises, relative to the wild-type murine (preferably murine) TCR β chain constant region, a substitution of an amino acid at position 3, e.g., R with K, a substitution of an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, a deletion of amino acids at positions 17, 21-25, and a mutation of an amino acid at position 56, e.g., serine S to cysteine ​​C; the modified TCR β chain constant region is derived from the constant region of a murine (preferably murine) TCR β chain and comprises, relative to the constant region of a wild-type murine (preferably murine) TCR β chain, a substitution of an amino acid at position 3, e.g., R, with K; a substitution of an amino acid at position 6, e.g., T, with F; a substitution of K at position 9 with E; a substitution of S at position 11 with A; a substitution of L at position 12 with V; a deletion of amino acids at positions 17, 21-25; a mutation of an amino acid at position 56, e.g., serine S, to cysteine ​​C; and a substitution of lysine at position 150, 168, or 170 with arginine; 17. The synthetic T cell receptor antigen receptor of claim 16, wherein the modified TCR β chain constant region is derived from the constant region of a murine (preferably murine) TCR β chain and comprises, relative to the constant region of a wild-type murine (preferably murine) TCR β chain, a substitution of an amino acid at position 3, e.g., R with K, a substitution of an amino acid at position 6, e.g., T with F, a substitution of K with E at position 9, a substitution of S with A at position 11, a substitution of L with V at position 12, a deletion of amino acids at positions 17, 21-25, and a substitution of lysine with arginine at position 150, 168 or 170.

19. 17. The synthetic T cell receptor antigen receptor of claim 16, wherein the second constant region comprises an amino acid sequence set forth in one of SEQ ID NOs: 2, 4, 6, 9, 17, 32, or 44.

20. 3. The synthetic T cell receptor antigen receptor of claim 1, wherein the first target binding region and the first constant region are linked directly or via a linker, and / or the second target binding region and the second constant region are linked directly or via a linker.

21. the linker is selected from a rigid linker, a flexible linker, a cleavable linker or a nonsense amino acid; Preferably, the amino acid sequence of the rigid linker is one or more selected from SEQ ID NO: 49 to 59; Preferably, said flexible linker is selected from glycine and / or serine rich peptide fragments, preferably said flexible linker is one or more selected from SEQ ID NO: 60-112; 21. The synthetic T cell receptor antigen receptor of claim 20, wherein the cleavable linker is preferably one or more selected from SEQ ID NOs: 113-117.

22. 1. A synthetic T cell receptor antigen receptor complex, comprising: A synthetic T cell receptor-antigen receptor complex, comprising the synthetic T cell receptor-antigen receptor of any one of claims 1 to 21, and CD3ε, CD3γ, CD3δ, and CD3ζ.

23. the CD3ε, CD3γ, CD3δ and / or CD3ζ are derived from a human; Preferably, the CD3ε comprises the amino acid sequence shown in SEQ ID NO: 20, Preferably, the CD3γ comprises the amino acid sequence shown in SEQ ID NO: 18, Preferably, the CD3δ comprises the amino acid sequence shown in SEQ ID NO: 19, 23. The synthetic T cell receptor antigen receptor complex of claim 22, wherein the CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

21.

24. An antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising CDR1-3; i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) an antibody or antigen-binding fragment, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:

38.

25. 25. The antibody or antigen-binding fragment of claim 24, wherein the antibody or antigen-binding fragment is a single-chain antibody or a single-domain antibody.

26. 25. The antibody or antigen-binding fragment of claim 24, wherein the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 28 or 29.

27. An antigen receptor, the antigen receptor comprises a transmembrane region, an intracellular region, and one or more identical or different extracellular binding domains, the extracellular binding domains being extracellular antigen-binding domains; the extracellular antigen-binding domain comprises CDR1-3; i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) An antigen receptor, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

28. The antigen receptor according to claim 27, wherein the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment according to any one of claims 24 to 26.

29. the antigen receptor is a STAR, a TCR, or a CAR; Preferably, the transmembrane region is derived from human CD8, The antigen receptor according to claim 27, wherein the intracellular region is preferably derived from 4-1BB, CD28, or CD3ζ.

30. 28. The antigen receptor of claim 27, wherein the transmembrane region and the one or more extracellular antigen-binding domains are directly linked or linked via a linker.

31. the linker is selected from a rigid linker, a flexible linker, a cleavable linker or a nonsense amino acid; Preferably, the amino acid sequence of the rigid linker is one or more selected from SEQ ID NO: 49 to 59; Preferably, said flexible linker is selected from glycine and / or serine rich peptide fragments, preferably said flexible linker is one or more selected from SEQ ID NO: 60-112; The antigen receptor according to claim 30, wherein the cleavable linker is preferably one or more selected from SEQ ID NOs: 113 to 117.

32. A nucleic acid encoding a synthetic T cell receptor-antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor-antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen-binding fragment according to any one of claims 24 to 26, or an antigen receptor according to any one of claims 27 to 31.

33. A vector comprising the nucleic acid of claim 32.

34. A host cell comprising a nucleic acid according to claim 32 or a vector according to claim 33.

35. An immune cell characterized by expressing a synthetic T cell receptor-antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor-antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen-binding fragment according to any one of claims 24 to 26, or an antigen receptor according to any one of claims 27 to 31.

36. 36. The immune cell of claim 35, comprising one or more nucleic acids of claim 32.

37. The immune cell of claim 35 or 36, which is selected from a T cell, a Treg cell, a macrophage, a NK cell, a NKT cell, a peripheral blood mononuclear cell, a TIL cell or a dendritic cell (DC).

38. 37. The immune cell of claim 35 or 36, isolated from a T cell of a subject.

39. A method for preparing immune cells, comprising transmitting the nucleic acid sequence according to claim 32 to immune cells to express the nucleic acid sequence, thereby obtaining immune cells.

40. Step 1) of screening positive T cells to obtain the nucleic acid of claim 32; 2) isolating and culturing primary T cells; and step 3) delivering the nucleic acid obtained in step 1) to the primary T cell described in step 2) to obtain a recombinant T cell that expresses the synthetic T cell receptor antigen receptor described in any one of claims 1 to 21.

41. (1) a step of screening positive T cells to obtain the nucleic acid of claim 32; Step (2) of ligating the nucleic acid obtained in step (1) into a vector backbone to obtain an expression vector; Step (3) of transforming the expression vector obtained in step (2) into a host cell and then inducing expression thereof; and (4) obtaining a synthetic T cell receptor antigen receptor.

42. Use of a synthetic T cell receptor antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen-binding fragment according to any one of claims 24 to 26, an antigen receptor according to any one of claims 27 to 31, a nucleic acid according to claim 32, or an immune cell according to any one of claims 35 to 38 in the preparation of a product for diagnosing or treating a tumor.

43. The tumors include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, malignant melanoma, myelodysplastic syndrome, and sarcoma; Preferably, said leukemia comprises acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; Preferably, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; Preferably, the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma; Preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia; 43. The use according to claim 42, wherein preferably said chronic myelogenous leukemia is chronic myelomonocytic leukemia.

44. A pharmaceutical composition comprising a synthetic T cell receptor-antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor-antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen-binding fragment according to any one of claims 24 to 26, an antigen receptor according to any one of claims 27 to 31, a nucleic acid according to claim 32, and an immune cell according to any one of claims 35 to 38.

45. A kit comprising a synthetic T cell receptor-antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor-antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen-binding fragment according to any one of claims 24 to 26, an antigen receptor according to any one of claims 27 to 31, a nucleic acid according to claim 32, and an immune cell according to any one of claims 35 to 38.

46. A method for treating a tumor, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment of any one of claims 24 to 26, the immune cell of any one of claims 35 to 38, or the pharmaceutical composition of claim 44.

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